Use of small molecule radioligands to discover inhibitors of amyloid-beta peptide production and for diagnostic imaging

ABSTRACT

This invention relates to a novel method of screening for inhibitors of beta-amyloid production, and thereby identifying such inhibitors as therapeutics for neurological and other disorders involving APP processing and beta-amyloid production. This invention also relates to identifying macromolecules involved in APP processing and beta-amyloid production. Furthermore, inhibitors identified by the screening method of the present invention are useful in the treatment of neurological disorders, such as Alzheimer&#39;s disease, which involve elevated levels of Aβ peptides.

This application is a continuation of U.S. patent application Ser. No.09/573,789 filed May 17, 2000.

FIELD OF THE INVENTION

This invention relates to a novel method of screening for inhibitors ofbeta-amyloid production, and thereby identifying such inhibitors astherapeutics for neurological and other disorders involving APPprocessing and beta-amyloid production. This invention also relates toidentifying macromolecules involved in APP processing and beta-amyloidproduction. Furthermore, inhibitors identified by the screening methodof the present invention are useful in the treatment of neurologicaldisorders, such as Alzheimer's disease, which involve elevated levels ofAβ peptides.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a degenerative brain disorder characterizedclinically by progressive loss of memory, temporal and localorientation, cognition, reasoning, judgment and emotional stability. ADis a common cause of progressive dementia in humans and is one of themajor causes of death in the United States. AD has been observed in allraces and ethnic groups worldwide, and is a major present and futurehealth problem. No treatment that effectively prevents AD or reversesthe clinical symptoms and underlying pathophysiology is currentlyavailable (for review, Dennis J. Selkoe; Cell Biology of the amyloid(beta)-protein precursor and the mechanism of Alzheimer's disease, AnnuRev Cell Biol, 1994, 10: 373-403).

Histopathological examination of brain tissue derived upon autopsy orfrom neurosurgical specimens in effected individuals revealed theoccurrence of amyloid plaques and neurofibrillar tangles in the cerebralcortex of such patients. Similar alterations were observed in patientswith Trisomy 21 (Down's syndrome), and hereditary cerebral hemorrhagewith amyloidosis of the Dutch-type. Neurofibrillar tangles arenonmembrane-bound bundles of abnormal proteinaceous filaments andbiochemical and immunochemical studies led to the conclusion that theirprinciple protein subunit is an altered phosphorylated form of the tauprotein (reviewed in Selkoe, 1994).

Biochemical and immunological studies revealed that the dominantproteinaceous component of the amyloid plaque is an approximately 4.2kilodalton (kD) protein of about 39 to 43 amino acids. This protein wasdesignated Aβ, β-amyloid peptide, and sometimes β/A4; referred to hereinas Aβ. In addition to deposition of Aβ in amyloid plaques, Aβ is alsofound in the walls of meningeal and.parenchymal arterioles, smallarteries, capillaries, and sometimes, venules. Aβ was first purified,and a partial amino acid reported, in 1984 (Glenner and Wong, Biochem.Biophys. Res. Commun. 120: 885-890). The isolation and sequence data forthe first 28 amino acids are described in U.S. Pat. No 4,666,829.

Compelling evidence accumulated during the last decade revealed that Aβis an internal polypeptide derived from a type 1 integral membraneprotein, termed β amyloid precursor protein (APP). β APP is normallyproduced by many cells both in vivo and in cultured cells, derived fromvarious animals and humans. Aβ is derived from cleavage of β APP by asyet unknown enzyme (protease) system(s), collectively termed secretases.

The existence of at least four proteolytic activities has beenpostulated. They include β secretase(s), generating the N-terminus ofAβ, α secretase(s) cleaving around the 16/17 peptide bond in Aβ, and γsecretases, generating C-terminal Aβ fragments ending at position 38,39, 40, 42, and 43 or generating C-terminal extended precursors whichare subsequently truncated to the above polypeptides.

The gene encoding a human aspartic protease that cleaves the β-secretasesite of β-amyloid precursor protein has recently been isolated; thisgene and encoded protein is designated as BACE (Vassar et al., Science(1999) 286: 735-741) or as memapsin-2 (Lin et al., PNAS (2000) 97:1456-1460) and is designated herein as “BACE/memapsin-2”.

Several lines of evidence suggest that abnormal accumulation of Aβ playsa key role in the pathogenesis of AD. Firstly, Aβ is the major proteinfound in amyloid plaques. Secondly, Aβ is neurotoxic and may be causallyrelated to neuronal death observed in AD patients. Thirdly, missense DNAmutations at position 717 in the 770 isoform of β APP can be found ineffected members but not unaffected members of several families with agenetically determined (familiar) form of AD. In addition, several otherβ APP mutations have been described in familial forms of AD. Fourthly,similar neuropathological changes have been observed in transgenicanimals overexpressing mutant forms of human β APP. Fifthly, individualswith Down's syndrome have an increased gene dosage of β APP and developearly-onset AD. Taken together, these observations strongly suggest thatβP depositions may be causally related to the AD.

It is hypothesized that inhibiting the production of Aβ will prevent andreduce neurological degeneration, by controlling the formation ofamyloid plaques, reducing neurotoxicity and, generally, mediating thepathology associated with Aβ production. One method of treatment methodswould therefore be based on drugs that inhibit the formation of Aβ invivo.

Methods of treatment could target the formation of Aβ through theenzymes involved in the proteolytic processing of β amyloid precursorprotein. Compounds that inhibit β or γ secretase activity, eitherdirectly or indirectly, could control the production of Aβ.Advantageously, compounds that specifically target γ secretases, couldcontrol the production of Aβ. Such inhibition of β or γ secretases couldthereby reduce production of Aβ, which, thereby, could reduce or preventthe neurological disorders associated with Aβ protein.

It is believed that several macromolecules, some of which haveproteolytic activity, are involved in the processing of amyloidprecursor protein (APP) . This processing leads to several productsincluding the β-amyloid peptides (Aβ) believed etiologically importantin Alzheimers Disease. We have discovered novel tagged compounds,functional in themselves as Aβ inhibitors, for use in identifying a siteor sites on one or more macromolecules critical to the processing of βAPP and the production of Aβ. We have discovered novel tagged compoundswhich inhibit the proteolytic activity leading to production of Aβ byinteracting with one or more macromolecules critical to the processingof APP and the production of Aβ. We have also discovered a site ofaction of these tagged compounds using radioisotope tagged derivativesof a compound of Formula (I).

Three examples of tagged compounds include (I-7T), (I-11T), and (I-43T):

-   -   (I-7) : R**=¹H; Y=—O—;    -   (I-7T): R**=³H; Y=—O—; and    -   (I-11): R**=¹H; Y=—C(═O)—;    -   (I-11T): R**=³H; Y=—C(═O)—; and    -   (I-43) : R**=¹H;    -   (I-43T): R**=³H.        The concentration of Compound (I-7) leading to half-maximal        inhibition (IC₅₀) of proteolytic activity leading to Aβ        production in HEK₂₉₃ cells expressing APP 695 wt is similar to        the concentration leading to half-maximal inhibition (IC₅₀) of        Compound (I-7T) binding to membranes derived from the same cell        line. The correlation holds for compounds (I-11T) and (I-43T).        Also using a compound of Formula (I), we have discovered a        macromolecule containing a binding site of action for compounds        of Formula (I) critical to the processing of APP and the        production of Aβ.

Furthermore, we have discovered through competitive binding studies thatthere is a good correlation between the ability of a series of compoundsto inhibit the proteolytic activity leading to production of Aβ and toinhibit the binding of Compound (I-7T), (I-11T), or (I-43T) to saidmembranes. Thus, the binding of Compound (I-7T), (I-11T), or (I-43T) torelevant tissues and cell lines, membranes derived from relevant tissuesand cell lines, as well as isolated macromolecules and complexes ofisolated macromolecules, is useful in the identification of inhibitorsof Aβ production through competitive binding assays. Furthermore, suchcompetitive binding assays are useful in identification of inhibitors ofproteolytic activity leading to Aβ production for the treatment ofAlzheimer's disease. Furthermore, such competitive binding assays areuseful in identification of inhibitors of proteolytic activity leadingto Aβ production for the treatment of neurological disorders and otherdisorders involving Aβ, APP, and/or Aβ/APP associated macromolecules,and other macromolecules associated with the site of Compound (I-7T),(I-11T), or (I-43T) binding.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a novel method ofscreening for inhibitors of beta-amyloid production, and therebyidentifying such inhibitors as therapeutics for neurological and otherdisorders involved in APP processing and beta-amyloid production. Themethod comprises 1) contacting a potential inhibitor of beta-amyloidproduction and a tagged inhibitor of beta-amyloid production with atleast one macromolecule involved in the processing of APP and theproduction of beta-amyloid peptide, said macromolecule containing abinding site specific for said tagged inhibitor of beta-amyloidproduction; 2) separating the tagged inhibitor of beta-amyloidproduction bound to said macromolecule from the tagged inhibitor ofbeta-amyloid production free from said macromolecule; and 3) determiningan inhibitory concentration of the potential inhibitor of beta-amyloidproduction from the concentration of tagged inhibitor of beta-amyloidproduction bound to said macromolecule.

It is another object of the present invention to provide the use of atagged inhibitor of beta-amyloid production to identify macromoleculesinvolved in APP processing.

It is another object of the present invention to provide themacromolecules involved in APP processing which a tagged inhibitor ofbeta-amyloid production binds to specifically.

It is another object of the present invention to provide the use ofmacromolecules involved in APP processing, which a tagged inhibitor ofbeta-amyloid production binds to specifically, for the identification ofinhibitors as therapeutics for neurological and other disorders involvedin APP processing and beta-amyloid production.

It is another object of the present invention to provide the use ofmacromolecules involved in APP processing, which a tagged inhibitor ofbeta-amyloid production binds to specifically, for the assaying ofinhibitors of beta-amyloid production.

It is another object of the present invention to provide pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and atherapeutically effective amount of an inhibitor of beta-amyloidproduction, or a pharmaceutically acceptable salt or prodrug formthereof, identified by the screening assay of the present invention.

It is another object of the present invention to provide a method fortreating degenerative neurological disorders involving beta-amyloidproduction, including Alzheimer's disease, comprising administering to ahost in need of such treatment a therapeutically effective amount of aninhibitor of beta-amyloid production, or a pharmaceutically acceptablesalt or prodrug form thereof, identified by the screening assay of thepresent invention.

It is another object of the present invention to provide an inhibitor ofbeta-amyloid production which interacts with the binding site for acompound of Formula (I-7T) on a macromolecule involved in the productionof beta-amyloid peptide.

These and other objects, which will become apparent during the followingdetailed description, have been achieved by the inventors' discoverythat compounds of Formula (I):

-   -   (I-7) R**=¹H; Y=—O—;    -   (I-7T): R**=³H; Y=—O—; and    -   (I-11): R**=¹H; Y=—C(═O)—;    -   (I-11T): R**=³H; Y=—C(═O)—; and    -   (I-43) R**=¹H;    -   (I-43T): R**=³H;        bind specifically to a binding site on a macromolecule or a        complex of macromolecules involved in APP processing to produce        reduction of Aβ peptide production. For example, the        concentration of Compound (I-7) leading to half-maximal        inhibition (IC₅₀) of Aβ production in HEK₂₉₃ cells expressing        APP 695 wt is similar to the concentration leading to        half-maximal inhibition (IC₅₀) of Compound (I-7T) binding to        membranes derived from the same cell line.

It is another object of the present invention to provide radiolabeledinhibitors of APP processing and/or the production of beta-amyloidproduction for use in methods of in vivo diagnostic imaging in thediagnosis of diseases involving APP processing and/or the production ofbeta-amyloid production. Also provided in the present invention aremethods of in vivo diagnostic imaging comprising administering to asubject a diagnostically effective amount of a radiolabeled inhibitor ofAPP processing and/or the production of beta-amyloid production

FIG. 1 illustrates the correlation between results of the RadioligandCompetition Binding Assay and the cross-linking assay of Example 103.

FIG. 2 illustrates a fluorography of a 12% SDS-PAGE afterimmunoprecipitation of specifically cross-linked polypepetides bypresenilin-1 antibodies.

FIG. 3 illustrates isolation of cross-linked polypeptides by presenilin1 affinity chromatography.

FIG. 4 illustrates a fluorography of a 12% SDS-PAGE afterimmunoprecipitation of specifically cross-linked polypepetides bypresenilin-2 antibodies.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[1] Thus, in a first embodiment, the present invention provides a methodof screening for inhibitors of beta-amyloid production comprising,

-   -   1) contacting a potential inhibitor of beta-amyloid production        and a tagged inhibitor of beta-amyloid production with at least        one macromolecule involved in the processing of APP and/or the        production of beta-amyloid peptide, said macromolecule        containing a binding site specific for said tagged inhibitor of        beta-amyloid production;    -   2) separating the tagged inhibitor of beta-amyloid production        bound to said macromolecule from the tagged inhibitor of        beta-amyloid production free from said macromolecule; and    -   3) determining an inhibitory concentration of the potential        inhibitor of beta-amyloid production from the concentration of        tagged inhibitor of beta-amyloid production bound to said        macromolecule.

[1a] The present invention provides the foregoing method wherein themacromolecule is selected from:

-   -   (1) presenilin-1;    -   (2) presenilin-2;    -   (3) β secretase;    -   (4) α secretase;    -   (5) γ secretase; or    -   (6) BACE/memapsin 2.

[2) In a more preferred embodiment the present invention provides amethod wherein the tagged inhibitor of beta-amyloid production comprisesa radiolabeled inhibitor of beta-amyloid production, a fluorescencelabeled inhibitor of beta-amyloid production or a biotin labeledinhibitor of beta-amyloid production.

[3] In a more preferred embodiment the tagged inhibitor of beta-amyloidproduction comprises a radiolabeled inhibitor of beta-amyloidproduction.

[4] In an even more preferred embodiment the present invention providesa method wherein the tagged inhibitor of beta-amyloid productioncomprises a tritium or iodine radiolabeled inhibitor of beta-amyloidproduction.

[5) In an even more preferred embodiment the present invention providesa method wherein the tagged inhibitor of beta-amyloid productioncomprises a tritium labeled inhibitor of beta-amyloid production.

[6] In an even more preferred embodiment the present invention, providesa method wherein the tagged inhibitor of beta-amyloid productioncomprises a compound of the Formula (I):

wherein:

-   -   at least one atom of the compound of the Formula (I) is        radiolabeled;    -   Q is —NR¹R²;    -   R¹, at each occurrence, is independently selected from: H;        -   C₁-C₆ alkyl substituted with 0-3 R^(1a);        -   C₃-C₁₀ carbocycle substituted with 0-3 R^(1b);        -   C₆-C₁₀ aryl substituted with 0-3 R^(1b); and        -   5 to 10 membered heterocycle substituted with 0-3 R^(1b);    -   R^(1a), at each occurrence, is independently selected from H,        C₁-C₆ alkyl, OR¹⁴, Cl, F, Br, I, ═O, CN, NO₂, NR¹⁵R¹⁶, phenyl,        CF₃;        -   C₃-C₁₀ carbocycle substituted with 0-3 R^(1b);        -   C₆-C₁₀ aryl substituted with 0-3 R^(1b); and        -   5 to 10 membered heterocycle substituted with 0-3 R^(1b);    -   R^(1b), at each occurrence, is independently selected from H,        OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,        or CF₃;    -   R² is independently selected from H, OH, C₁-C₆ alkyl, C₁-C₆        alkoxy, C₃-C₁₀ carbocycle, C₆-C₁₀ aryl and 5 to 10 membered        heterocycle;    -   R³ is C₁-C₆ alkyl substituted with 0-1 R⁴;    -   R⁴ is H, OH, C₁-C₆ alkoxy, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, C₃-C₁₀ carbocycle, C₆-C₁₀ aryl, or 5 to 10 membered        heterocycle;    -   R⁵ is H, OR¹⁴;        -   C₁-C₆ alkyl substituted with 0-3 R^(5b);        -   C₁-C₆ alkoxy substituted with 0-3 R^(5b);        -   C₂-C₆ alkenyl substituted with 0-3 R^(5b);        -   C₂-C₆ alkynyl substituted with 0-3 R^(5b);        -   C₃-C₁₀ carbocycle substituted with 0-3 R^(5c);        -   C₆-C₁₀ aryl substituted with 0-3 R^(5c); or        -   5 to 10 membered heterocycle substituted with 0-3 R^(5c);    -   R^(5b), at each occurrence, is independently selected from:        -   H, C₁-C₆ alkyl, CF₃, OR¹⁴, Cl, F, Br, I, ═O, CN, NO₂,            NR¹⁵R¹⁶;        -   C₃-C₁₀ carbocycle substituted with 0-3 R^(5c);        -   C₆-C₁₀ aryl substituted with 0-3 R^(5c); or        -   5 to 10 membered heterocycle substituted with 0-3 R^(5c);    -   R^(5c), at each occurrence, is independently selected from H,        OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,        or CF₃;    -   R⁶ is H;        -   C₁-C₆ alkyl substituted with 0-3 R^(6a);        -   C₃-C₁₀ carbocycle substituted with 0-3 R^(6b); or        -   C₆-C₁₀ aryl substituted with 0-3R^(6b);    -   R^(6a), at each occurrence, is independently selected from H,        C₁-C₆ alkyl, OR¹⁴, Cl, F, Br, I, ═O, CN, NO₂, NR¹⁵R¹⁶, phenyl or        CF₃;    -   R^(6b), at each occurrence, is independently selected from H,        OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,        or CF₃;    -   W is —(CR⁸R^(8a))_(p)—;    -   p is 0 to 4;    -   R⁸ and R^(8a), at each occurrence, are independently selected        from H, C₁-C₄ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl and C₃-C₈        cycloalkyl;    -   X is a bond;        -   C₆-C₁₀ aryl substituted with 0-3 R^(Xb);        -   C₃-C₁₀ carbocycle substituted with 0-3 R^(Xb); or        -   5 to 10 membered heterocycle substituted with 0-3 R^(Xb);    -   R^(Xb), at each occurrence, is independently selected from H,        OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,        or CF₃;    -   Y is a bond or —(CR⁹R^(9a))_(t)—V—(CR⁹R^(9a))_(u)—;    -   t is 0 to 3;    -   u is 0 to 3;    -   R⁹ and R^(9a), at each occurrence, are independently selected        from H, C₁-C₆ alkyl or C₃-C₈ cycloalkyl;    -   V is a bond, —C(═O)—, —O—, —S—, —S(═O)—, —S(═O)₂—, —N(R¹⁹)—,        —C(═O)NR^(19b)—, —NR^(19b)C(═O)—, —NR^(19b)S(═O)₂—,        —S(═O)₂NR^(19b)—, —NR^(19b)S(═O)—, —S(═O)NR^(19b)—, —C(═O)O—, or        —OC(═O)—;    -   Z is H;        -   C₁-C₈ alkyl substituted with 0-2 R¹²;        -   C₂-C₄ alkenyl substituted with 0-2 R¹²;        -   C₂-C₄ alkynyl substituted with 0-2 R¹²;        -   C₆-C₁₀ aryl substituted with 0-4 R^(12b);        -   C₃-C₁₀ carbocycle substituted with 0-4 R^(12b); or        -   5 to 10 membered heterocycle substituted with 0-3 R^(12b);    -   R¹² is C₆-C₁₀ aryl substituted with 0-4 R^(12b);        -   C₃-C₁₀ carbocycle substituted with 0-4 R^(12b); or        -   5 to 10 membered heterocycle substituted with 0-3 R^(12b);    -   R^(12b), at each occurrence, is independently selected from H,        OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,        or CF₃;    -   B is a 5 to 10 membered lactam, wherein the lactam is saturated,        partially saturated or unsaturated; wherein each additional        lactam carbon is substituted with 0-2 R¹¹; and, optionally, the        lactam contains a heteroatom selected from —O—, —S—, —S(═O)—,        —S(═O)₂—, —N═ and —N(R¹⁰)—;    -   R¹⁰ is H, C(═O)R¹⁷, C(═O)OR¹⁷, C(═O)NR¹⁸R¹⁹,        -   S(═O)₂NR¹⁸R¹⁹, S(═O)₂R¹⁷;        -   C₁-C₆ alkyl optionally substituted with R^(10a);        -   C₆-C₁₀ aryl substituted with 0-4 R^(10b);        -   C₃-C₁₀ carbocycle substituted with 0-3 R^(10b); or        -   5 to 10 membered heterocycle optionally substituted with 0-3            R^(10b);    -   R^(10a), at each occurrence, is independently selected from H,        C₁-C₆ alkyl, C₃-C₆ cycloalkyl, OR¹⁴, Cl, F, Br, I, ═O, CN, NO₂,        NR¹⁵R¹⁶, phenyl or CF₃;    -   R^(10b), at each occurrence, is independently selected from H,        OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,        or CF₃;    -   R¹¹ is C₁-C₄ alkoxy, Cl, F, Br, I, ═O, CN, NO₂, NR¹⁸R¹⁹,        C(═O)R¹⁷, C(═O)OR¹⁷, C(═O)NR¹⁸R¹⁹, S(═O)₂NR¹⁸R¹⁹, CF₃;        -   C₁-C₆ alkyl optionally substituted with R^(11a);        -   C₆-C₁₀ aryl substituted with 0-3 R^(11b);        -   C₃-C₁₁ carbocycle substituted with 0-3 R^(11b); or        -   5 to 10 membered heterocycle substituted with 0-3 R^(11b);    -   alternatively, two R¹¹ substituents on the same carbon atoms may        be combined to form a C₃-C₆ carbocycle;    -   alternatively, two R¹¹ substituents on adjacent carbon atoms may        be combined to form a C₃-C₆ carbocycle or a benzo fused radical,        wherein said benzo fused radical is substituted with 0-3 R¹³;    -   R^(11a), at each occurrence, is independently selected from H,        C₁-C₆ alkyl, OR¹⁴, Cl, F, Br, I, ═O, CN, NO₂, NR¹⁵R¹⁶, phenyl or        CF₃;    -   R^(11b), at each occurrence, is independently selected from H,        OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,        or CF₃;    -   R¹³, at each occurrence, is independently selected from H, OH,        C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶, or        CF₃;    -   R¹⁴ is H, phenyl, benzyl, C₁-C₆ alkyl, or C₂-C₆ alkoxyalkyl;    -   R¹⁵, at each occurrence, is independently selected from H, C₁-C₆        alkyl, benzyl, phenethyl, —C(═O)—(C₁-C₆ alkyl) and        —S(═O)₂—(C₁-C₆ alkyl);    -   R¹⁶, at each occurrence, is independently selected from H, OH,        C₁-C₆ alkyl, benzyl, phenethyl, —C(═O)—(C₁-C₆ alkyl) and        —S(═O)₂—(C₁-C₆ alkyl);    -   R¹⁷ is H, phenyl, benzyl, C₁-C₆ alkyl, or C₂-C₆ alkoxyalkyl;    -   R¹⁸, at each occurrence, is independently selected from H, C₁-C₆        alkyl, benzyl, phenethyl, —C(═O)—(C₁-C₆ alkyl) and        —S(═O)₂—(C₁-C₆ alkyl); and    -   R¹⁹, at each occurrence, is independently selected from H, OH,        C₁-C₆ alkyl, phenyl, benzyl, phenethyl, —C(═O)—(C₁-C₆ alkyl) and        —S(═O)₂—(C₁-C₆ alkyl);    -   R^(19b) is H, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, phenyl, benzyl or        phenethyl; and    -   R²⁰ is H or C₁-C₆ alkyl.

[ ] In an even further more preferred embodiment the present invention,provides a method wherein Q of a compound of Formula (I) is —NH₂.

[7] In an even further more preferred embodiment the present invention,provides a method wherein R³ of a compound of Formula (I) is C₃-C₆alkyl.

[8] In an even further more preferred embodiment the present invention,provides a method wherein R³ of a compound of Formula (I) is C₃-C₆ alkylsubstituted with about 1 to about 4 ³H;

[ ] In an even further more preferred embodiment the present invention,provides a method wherein Q is —NH₂, and R³ is C₃-C₆ alkyl substitutedwith about 1 to about 4 ³H.

[9] In an even further more preferred embodiment the present invention,provides a method wherein the tagged inhibitor of beta-amyloidproduction comprises a compound of the Formula (II):

wherein:

-   -   at least one atom of the compound of the Formula (II) is        radiolabeled.

[10] In an even further more preferred embodiment the present invention,provides a method wherein R³, in a compound of Formula (II), is C₃-C₆alkyl substituted with about 1 to about 4 ³H.

[11] In a most preferred embodiment the present invention, provides amethod wherein the tagged inhibitor of beta-amyloid production comprisesa compound of Formula:

wherein m is about 2.

[12] In a further most preferred embodiment the present invention,provides a method wherein the tagged inhibitor of beta-amyloidproduction comprises a compound of Formula (I-43T)

wherein m is about 2.

[ ] In yet another preferred embodiment the present invention provides amethod wherein the tagged inhibitor of beta-amyloid production comprisesa compound selected from U.S. Pat. No. 5,703,129; PCT applicationWO98/28268; PCT application WO98/22441; PCT application WO98/22433; PCTapplication WO98/22430; PCT application WO98/22493; PCT applicationWO98/22494; PCT application WO98/38177; or PCT application WO95/09838;wherein the compound has been tagged for purposes of the invention.

[13] In another preferred embodiment the present invention provides amethod wherein at least one macromolecule involved in the processing ofAPP and the production of beta-amyloid peptide comprises presenilin 1 ora fragment of presenilin 1.

[14] In another preferred embodiment the present invention provides amethod wherein the macromolecule involved in the processing of APPand/or the production of beta-amyloid peptide comprises:

-   -   (1) presenilin-1;    -   (2) presenilin-2;    -   (3) β secretase;    -   (4) α secretase;    -   (5) γ secretase; or    -   (6) BACE/memapsin 2;        or any fragment or derivative thereof.

[ ] In another preferred embodiment the present invention provides amethod wherein at least one macromolecule involved in the processing ofAPP and the production of beta-amyloid peptide comprises either 1)presenilin 1 or a fragment of presenilin 1 or 2) presenilin 2 or afragment of presenilin 2; but not both.

[15] In yet another preferred embodiment the present invention providesa method wherein the inhibitory concentration is half maximal inhibitoryconcentration.

[16] In a second embodiment, the present invention provides apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of an inhibitor ofbeta-amyloid production identified by the screening assay of claim 1 ora pharmaceutically acceptable salt or prodrug form thereof.

[17] In a third embodiment, the present invention provides a method fortreating degenerative neurological disorders involving beta-amyloidproduction comprising administering to a host in need of such treatmenta therapeutically effective amount of an inhibitor of beta-amyloidproduction identified by the screening assay of claim 1 or apharmaceutically acceptable salt or prodrug form thereof.

[18] In a preferred third embodiment the degenerative neurologicaldisorder is Alzheimer's Disease.

[19] In a fourth embodiment, the present invention provides a method ofidentifying a macromolecule involved in APP processing comprising

-   -   1) contacting a tagged inhibitor of beta-amyloid production with        material suspected to contain a macromolecule involved in APP        processing;    -   2) separating a complex comprising a tagged inhibitor of        beta-amyloid production and a macromolecule involved in APP        processing; and    -   3) identifying the complex.

[20] In a preferred fourth embodiment the present invention provides amethod wherein the tagged inhibitor of beta-amyloid production comprisesa radiolabeled inhibitor of beta-amyloid production, a fluorescencelabeled inhibitor of beta-amyloid production, a biotin labeled inhibitorof beta-amyloid production, a photoaffinity labeled inhibitor ofbeta-amyloid production, or any combination of tags thereof in oneinhibitor of beta-amyloid production.

[21] In a preferred fourth embodiment the present invention provides amethod wherein the tagged inhibitor of beta-amyloid production comprisesa radiolabeled inhibitor of beta-amyloid production.

[22] In a more preferred fourth embodiment the present inventionprovides a method wherein the tagged inhibitor of beta-amyloidproduction comprises a tritium labeled inhibitor of beta-amyloidproduction.

[23] In a more preferred fourth embodiment the present inventionprovides a method wherein the tagged inhibitor of beta-amyloidproduction comprises a compound of Formula (I):

wherein:

-   -   at least one atom of the compound of the Formula (I) is        radiolabeled;    -   Q is —NR¹R²;    -   R¹, at each occurrence, is independently selected from: H;        -   C₁-C₆ alkyl substituted with 0-3 R^(1a);        -   C₃-C₁₀ carbocycle substituted with 0-3 R^(1b);        -   C₆-C₁₀ aryl substituted with 0-3 R^(1b); and        -   5 to 10 membered heterocycle substituted with 0-3 R^(1b);    -   R^(1a), at each occurrence, is independently selected from H,        C₁-C₆ alkyl, OR¹⁴, Cl, F, Br, I, ═O, CN, NO₂, NR¹⁵R¹⁶, phenyl,        CF₃;        -   C₃-C₁₀ carbocycle substituted with 0-3 R^(1b);        -   C₆-C₁₀ aryl substituted with 0-3 R^(1b); and        -   5 to 10 membered heterocycle substituted with 0-3 R^(1b);    -   R^(1b), at each occurrence, is independently selected from H,        OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,        or CF₃;    -   R² is independently selected from H, OH, C₁-C₆ alkyl, C₁-C₆        alkoxy, C₃-C₁₀ carbocycle, C₆-C₁₀ aryl and 5 to 10 membered        heterocycle;    -   R³ is C₁-C₆ alkyl substituted with 0-1 R⁴;    -   R⁴ is H, OH, C₁-C₆ alkoxy, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, C₃-C₁₀ carbocycle, C₆-C₁₀ aryl, or 5 to 10 membered        heterocycle;    -   R⁵ is H, OR¹⁴;        -   C₁-C₆ alkyl substituted with 0-3 R^(5b);        -   C₁-C₆ alkoxy substituted with 0-3 R^(5b);        -   C₂-C₆ alkenyl substituted with 0-3 R^(5b);        -   C₂-C₆ alkynyl substituted with 0-3 R^(5b);        -   C₃-C₁₀ carbocycle substituted with 0-3 R^(5c);        -   C₆-C₁₀ aryl substituted with 0-3 R^(5c); or        -   5 to 10 membered heterocycle substituted with 0-3 R^(5c);    -   R^(5b), at each occurrence, is independently selected from:        -   H, C₁-C₆ alkyl, CF₃, OR¹⁴, Cl, F, Br, I, ═O, CN, NO₂,            NR¹⁵R¹⁶;        -   C₃-C₁₀ carbocycle substituted with 0-3 R^(5c);        -   C₆-C₁₀ aryl substituted with 0-3 R^(5c); or        -   5 to 10 membered heterocycle substituted with 0-3 R^(5c);    -   R^(5c), at each occurrence, is independently selected from H,        OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,        or CF₃;    -   R⁶ is H;        -   C₁-C₆ alkyl substituted with 0-3 R^(6a);        -   C₃-C₁₀ carbocycle substituted with 0-3 R^(6b); or        -   C₆-C₁₀ aryl substituted with 0-3R^(6b);    -   R^(6a), at each occurrence, is independently selected from H,        C₁-C₆ alkyl, OR¹⁴, Cl, F, Br, I, ═O, CN, NO₂, NR¹⁵R¹⁶, phenyl or        CF₃;    -   R^(6b), at each occurrence, is independently selected from H,        OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,        or CF₃;    -   W is —(CR⁸R^(8a))_(p)—;    -   p is 0 to 4;    -   R⁸ and R⁸a, at each occurrence, are independently selected from        H, C₁-C₄ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl and C₃-C₈        cycloalkyl;    -   X is a bond;        -   C₆-C₁₀ aryl substituted with 0-3 R^(Xb);        -   C₃-C₁₀ carbocycle substituted with 0-3 R^(Xb); or        -   5 to 10 membered heterocycle substituted with 0-3 R^(Xb);    -   R^(Xb), at each occurrence, is independently selected from H,        OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,        or CF₃;    -   Y is a bond or —(CR⁹R^(9a))_(t)—V—(CR⁹R^(9a))_(u)—;    -   t is 0 to 3;    -   u is 0 to 3;    -   R⁹ and R^(9a), at each occurrence, are independently selected        from H, C₁-C₆ alkyl or C₃-C₈ cycloalkyl;    -   V is a bond, —C(═O)—, —O—, —S—, —S(═O)—, —S(═O)₂—, —N(R¹⁹)—,        —C(═O)NR^(19b)—, —NR^(19b)C(═O)—, —NR^(19b)S(═O)₂—,        —S(═O)₂NR¹⁹—, —NR^(19b)S(═O)—, —S(═O)NR^(19b)—, —C(═O)O—, or        —OC(═O)—;    -   Z is H;        -   C₁-C₈ alkyl substituted with 0-2 R¹²;        -   C₂-C₄ alkenyl substituted with 0-2 R¹²;        -   C₂-C₄ alkynyl substituted with 0-2 R¹²;        -   C₆-C₁₀ aryl substituted with 0-4 R^(12b);        -   C₃-C₁₀ carbocycle substituted with 0-4 R^(12b); or        -   5 to 10 membered heterocycle substituted with 0-3 R^(12b);    -   R¹² is C₆-C₁₀ aryl substituted with 0-4 R^(12b);        -   C₃-C₁₀ carbocycle substituted with 0-4 R^(12b); or        -   5 to 10 membered heterocycle substituted with 0-3 R^(12b);    -   R^(12b), at each occurrence, is independently selected from H,        OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,        or CF₃;    -   B is a 5 to 10 membered lactam, wherein the lactam is saturated,        partially saturated or unsaturated; wherein each additional        lactam carbon is substituted with 0-2 R¹¹; and, optionally, the        lactam contains a heteroatom selected from —O—, —S—, —S(═O)—,        —S(═O)₂—, —N═, and —N(R¹⁰) ;    -   R¹⁰ is H, C(═O)R¹⁷, C(═O)OR¹⁷, C(═O)NR¹⁸R¹⁹, S(═O)₂NR¹⁸R¹⁹, S        (═O)₂R¹⁷;        -   C₁-C₆ alkyl optionally substituted with R^(10a);        -   C₆-C₁₀ aryl substituted with 0-4 R^(10b);        -   C₃-C₁₀ carbocycle substituted with 0-3 R^(10b); or        -   5 to 10 membered heterocycle optionally substituted with 0-3            R^(10b);    -   R^(10a), at each occurrence, is independently selected from H,        C₁-C₆ alkyl, C₃-C₆ cycloalkyl, OR¹⁴, Cl, F, Br, I, ═O, CN, NO₂,        NR¹⁵R¹⁶, phenyl or CF₃;    -   R^(10b), at each occurrence, is independently selected from H,        OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,        or CF₃;    -   R¹¹ is C₁-C₄ alkoxy, Cl, F, Br, I, ═O, CN, NO₂, NR¹⁸R¹⁹,        C(═O)R¹⁷, C(═O)OR¹⁷, C(═O)NR¹⁸R¹⁹, S(═O)₂NR¹⁸R¹⁹, CF₃;        -   C₁-C₆ alkyl optionally substituted with R^(11a);        -   C₆-C₁o aryl substituted with 0-3 R^(11b);        -   C₃-C₁o carbocycle substituted with 0-3 R^(11b); or        -   5 to 10 membered heterocycle substituted with 0-3 R^(11b);    -   alternatively, two R¹¹ substituents on the same carbon atoms may        be combined to form a C₃-C₆ carbocycle;    -   alternatively, two R¹¹ substituents on adjacent carbon atoms may        be combined to form a C₃-C₆ carbocycle or a benzo fused radical,        wherein said benzo fused radical is substituted with 0-3 R¹³;    -   R^(11a), at each occurrence, is independently selected from H,        C₁-C₆ alkyl, OR¹⁴, Cl, F, Br, I, ═O, CN, NO₂, NR¹⁵R¹⁶, phenyl or        CF₃;    -   R^(11b), at each occurrence, is independently selected from H,        OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,        or CF₃;    -   R¹³, at each occurrence, is independently selected from H, OH,        C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶, or        CF₃;    -   R¹⁴ is H, phenyl, benzyl, C₁-C₆ alkyl, or C₂-C₆ alkoxyalkyl;    -   R¹⁵, at each occurrence, is independently selected from H, C₁-C₆        alkyl, benzyl, phenethyl, —C(═O)—(C₁-C₆ alkyl) and        —S(═O)₂—(C₁-C₆ alkyl);    -   R¹⁶, at each occurrence, is independently selected from H, OH,        C₁-C₆ alkyl, benzyl, phenethyl, —C(═O)—(C₁-C₆ alkyl) and        —S(═O)₂—(C₁-C₆ alkyl);    -   R¹⁷ is H, phenyl, benzyl, C₁-C₆ alkyl, or C₂-C₆ alkoxyalkyl;    -   R¹⁸, at each occurrence, is independently selected from H, C₁-C₆        alkyl, benzyl, phenethyl, —C(═O)—(C₁-C₆ alkyl) and        —S(═O)₂—(C₁-C₆ alkyl); and    -   R¹⁹, at each occurrence, is independently selected from H, OH,        C₁-C₆ alkyl, phenyl, benzyl, phenethyl, —C(═O)—(C₁-C₆ alkyl) and        —S(═O)₂—(C₁-C₆ alkyl);    -   R^(19b) is H, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, phenyl, benzyl or        phenethyl; and    -   R²⁰ is H or C₁-C₆ alkyl.

[24] In an even more preferred fourth embodiment the present inventionprovides a method wherein the tagged inhibitor of beta-amyloidproduction comprises a compound of the Formula (I-7T):

wherein m is about 2.

[26] In another preferred fourth embodiment the present inventionprovides a method wherein the tagged inhibitor of beta-amyloidproduction is radiolabeled and photoaffinity labeled.

[27] In a more preferred fourth embodiment the present inventionprovides a method wherein the tagged inhibitor of beta-amyloidproduction comprises a compound of the Formula (I-11T):

wherein m is about 2.

[ ] In an even further more preferred fourth embodiment the presentinvention provides a method wherein the tagged inhibitor of beta-amyloidproduction comprises a compound of the Formula (I-43T):

wherein m is about 2.

[28] In fifth embodiment the present invention provides a macromoleculeinvolved in APP processing which a tagged inhibitor of beta-amyloidproduction binds to specifically.

[29] In a preferred fifth embodiment the present invention provides amacromolecule wherein the the tagged inhibitor of beta-amyloidproduction comprises a radiolabeled inhibitor of beta-amyloidproduction, a fluorescence labeled inhibitor of beta-amyloid production,a biotin labeled inhibitor of beta-amyloid production, a photoaffinitylabeled inhibitor of beta-amyloid production, or any combination of tagsthereof in one inhibitor of beta-amyloid production.

[30] In a preferred fifth embodiment the present invention provides amacromolecule wherein the tagged inhibitor of beta-amyloid productioncomprises a radiolabeled inhibitor of beta-amyloid production.

[31] In a more preferred fifth embodiment the present invention providesa macromolecule wherein the tagged inhibitor of beta-amyloid productioncomprises a compound of the Formula (I-7T):

wherein m is about 2.

[32] In another preferred fifth embodiment the present inventionprovides a macromolecule wherein the tagged inhibitor of beta-amyloidproduction comprises a compound of the Formula (I-11T):

wherein m is about 2.

[33] In another preferred fifth embodiment the present inventionprovides a macromolecule wherein the tagged inhibitor of beta-amyloidproduction comprises a compound of the Formula (I-43T):

wherein m is about 2.

[34] In another preferred fifth embodiment the present inventionprovides a macromolecule involved in APP processing which macromoleculeis presenilin 1 or a fragment of presenilin 1.

[35] In another preferred fifth embodiment the present inventionprovides a macromolecule involved in APP processing which macromoleculeis presenilin 2 or a fragment of presenilin 2.

[36] In a sixth embodiment the present invention provides an inhibitorof beta-amyloid production comprising a compound which interacts with abinding site on a macromolecule involved in the production ofbeta-amyloid peptide; wherein said binding site is identified by acompound of Formula (I-7T) or (I-43T):

wherein m is about 2.

[36] In the sixth embodiment the binding site is identified as aspecific binding site for a compound of Formula (I-7T) or (I-43T),wherein m is about 2.

[37] In a preferred sixth embodiment the macromolecule involved in theproduction of beta-amyloid peptide is presenilin 1 or a fragment ofpresenilin 1.

[38] In a preferred sixth embodiment the macromolecule involved in theproduction of beta-amyloid peptide is presenilin 2 or a fragment ofpresenilin 2.

[39] In another preferred sixth embodiment the invention provides aninhibitor of beta-amyloid production comprising a compound whichinteracts with a binding site on a macromolecule involved in theproduction of beta-amyloid peptide; wherein said binding site is aspecific binding site for a compound of Formula (I-7T), wherein m isabout 2; and the compound demonstrates a half maximal inhibitoryconcentration less than 10 micromolar for beta-amyloid production.

[40] In a more preferred sixth embodiment the invention provides aninhibitor of beta-amyloid production comprising a compound whichinteracts with a binding site on presenilin 1 or a fragment ofpresenilin 1; wherein said binding site is a specific binding site for acompound of Formula (I-7T), wherein m is about 2; and the compounddemonstrates a half maximal inhibitory concentration less than 10micromolar for beta-amyloid production.

[41] In another preferred sixth embodiment the invention provides aninhibitor of beta-amyloid production comprising a compound whichinteracts with a binding site on a macromolecule involved in theproduction of beta-amyloid peptide; wherein said binding site is aspecific binding site for a compound of Formula (I-43T), wherein m isabout 2; and the compound demonstrates a half maximal inhibitoryconcentration less than 10 micromolar for beta-amyloid production.

[42] In another more preferred sixth embodiment the invention providesan inhibitor of beta-amyloid production comprising a compound whichinteracts with a binding site on presenilin 1 or a fragment ofpresenilin 1; wherein said binding site is a specific binding site for acompound of Formula (I-43T), wherein m is about 2; and the compounddemonstrates a half maximal inhibitory concentration less than 10micromolar for beta-amyloid production.

[43] In a seventh embodiment the present invention provides a taggedinhibitor of beta-amyloid production comprising a tagged compound whichinteracts with a binding site on a macromolecule involved in theproduction of beta-amyloid peptide; wherein said binding site isidentified by a compound of Formula (I-7T):

wherein m is about 2;

[43] In the seventh embodiment the binding site is identified as aspecific binding site for a compound of Formula (I-7T) or (I-43T),wherein m is about 2.

[44] In a preferred seventh embodiment the macromolecule involved in theproduction of beta-amyloid peptide is presenilin 1 or a fragment ofpresenilin 1.

[45] In a preferred seventh embodiment the macromolecule involved in theproduction of beta-amyloid peptide is presenilin 2 or a fragment ofpresenilin 2.

[46] In another preferred seventh embodiment the invention provides atagged inhibitor of beta-amyloid production comprising a tagged compoundwhich interacts with a binding site on a macromolecule involved in theproduction of beta-amyloid peptide; wherein said binding site is aspecific binding site for a compound of Formula (I-7T), wherein m isabout 2; and the tagged compound demonstrates a half maximal inhibitoryconcentration less than 10 micromolar for beta-amyloid production.

[47] In a more preferred seventh embodiment the invention provides atagged inhibitor of beta-amyloid production comprising a tagged compoundwhich interacts with a binding site on presenilin 1 or a fragment ofpresenilin 1; wherein said binding site is a specific binding site for acompound of Formula (I-7T), wherein m is about 2; and the taggedcompound demonstrates a half maximal inhibitory concentration less than10 micromolar for beta-amyloid production.

[48] In another preferred seventh embodiment the invention provides atagged inhibitor of beta-amyloid production comprising a tagged compoundwhich interacts with a binding site on a macromolecule involved in theproduction of beta-amyloid peptide; wherein said binding site is aspecific binding site for a compound of Formula (I-43T), wherein m isabout 2; and the tagged compound demonstrates a half maximal inhibitoryconcentration less than 10 micromolar for beta-amyloid production.

[49] In another more preferred seventh embodiment the invention providesa tagged inhibitor of beta-amyloid production comprising a taggedcompound which interacts with a binding site on presenilin 1 or afragment of presenilin 1; wherein said binding site is a specificbinding site for a compound of Formula (I-43T), wherein m is about 2;and the tagged compound demonstrates a half maximal inhibitoryconcentration less than 10 micromolar for beta-amyloid production.

[ ] In yet another preferred embodiment the present invention provides atagged inhibitor of beta-amyloid production comprising a compoundselected from U.S. Pat. No. 5,703,129; PCT application WO98/28268; PCTapplication WO98/22441; PCT application WO98/22433; PCT applicationWO98/22430; PCT application WO98/22493; PCT application WO98/22494; PCTapplication WO98/38177; or PCT application WO95/09838; wherein thecompound has been tagged for purposes of the invention.

[ ] In an eighth embodiment the present invention provides a use of amacromolecule or complex of macromolecules involved in APP processing,which a tagged inhibitor of beta-amyloid production binds tospecifically, for the identification or assaying of inhibitors astherapeutics for neurological and other disorders involved in APPprocessing and beta-amyloid production.

[ ] In a preferred eighth embodiment the present invention provides ause of a macromolecule or complex of macromolecules involved in APPprocessing, which macromolecule or complex of macromolecules ispresenilin 1 or a fragment of presenilin 1.

[50] In a more preferred eighth embodiment the present inventionprovides a method of identifying inhibitors as therapeutics forneurological and other disorders involved in APP processing andbeta-amyloid production comprising

-   -   (1) contacting at least one macromolecule involved in APP        processing and beta-amyloid production, which macromolecule a        tagged inhibitor of beta-amyloid production binds to        specifically, with a potential inhibitor of beta-amyloid        production; and    -   (2) determining the level of inhibition of APP processing and        beta-amyloid production.

[51] In an even more preferred eighth embodiment the present inventionprovides a method wherein the macromolecule is a complex ofmacromolecules.

[52] In an even more preferred eighth embodiment the present inventionprovides a method of wherein the macromolecule is presenilin 1 or afragment of presenilin

[53] In an even more preferred eighth embodiment the present inventionprovides a method of wherein the macromolecule is presenilin 2 or afragment of presenilin 2.

[54] In a ninth embodiment the present invention provides a method oftreating Alzheimer's disease comprising administering to a host in needof such treatment a therapeutically effective amount of an inhibitor ofbeta-amyloid production, or a pharmaceutically acceptable salt orprodrug form thereof, wherein said inhibitor of beta-amyloid productionbinds to a binding site on a macromolecule involved in the production ofbeta-amyloid peptide and effects a decrease in production ofbeta-amyloid peptide;

-   -   wherein said binding site is a specific binding site for a        compound of Formula (I-7T) or (I-43T) wherein m is about 2.

[55] In a preferred ninth embodiment the macromolecule comprisespresenilin-1, a fragment of presenilin-1, presenilin-2, or a fragment ofpresenilin-2.

[56] In another preferred ninth embodiment the binding site is aspecific binding site for a compound of Formula (I-43T) wherein m isabout 2.

[57] In a more preferred ninth embodiment the macromolecule comprisespresenilin-1 or a fragment of presenilin-1.

[58] In another more preferred ninth embodiment the macromoleculecomprises presenilin-2 or a fragment of presenilin-2.

[ ] The present invention also provides radiolabeled inhibitors of APPprocessing and/or the production of beta-amyloid production for use inmethods of in vivo diagnostic imaging in the diagnosis of diseasesinvolving APP processing and/or the production of beta-amyloidproduction. Also provided in the present invention are methods of invivo diagnostic imaging comprising administering to a subject adiagnostically effective amount of a radiolabeled inhibitor of APPprocessing and/or the production of beta-amyloid production. As usedherein, the term “radiolabeled inhibitors of APP processing” or“radiolabeled inhibitor of beta-amyloid production”, when applied touses for in vivo diagnostic imaging, also includes any ligand that bindsto a macromolecule involved in APP processing or beta-amyloid productionwith an affinity and selectivity suitable for in vivo diagnosticimaging. Thus, such suitable ligand does not need to be an inhibitor ofAPP processing or beta-amyloid production. Although the radiolabeledligand may be an inhibitor of beta-amyloid production when administeredat therapeutic doses, the relatively low levels of radiolabeled ligandused in an in vivo diagnostic imaging procedure will generally be wellbelow such therapeutic levels. Thus, for purposes of in vivo diagnosticimaging, the term “radiolabeled inhibitor of APP processing” or“radiolabeled inhibitor of beta-amyloid production” refers to anysuitable radiolabeled compound which binds in vivo to the samemacromolecule target as a compound that is an inhibitor of APPprocessing and/or beta-amyloid production as described herein.

[59] The present invention includes the above-described method of invivo diagnostic imaging comprising administering to a subject adiagnostically effective amount of a radiolabeled inhibitor ofbeta-amyloid production.

[60] The present invention includes the above-described method of invivo diagnostic imaging wherein said method is used in the diagnosis ofa neurological disease which involves APP processing or elevated levelsof beta-amyloid, or both.

[61] The present invention includes the above-described method of invivo diagnostic imaging wherein said method is used in the diagnosis ofAlzheimer's disease.

[62] The present invention includes the above-described method of invivo diagnostic imaging wherein the radiolabeled inhibitor is suitablefor imaging of the brain of the subject.

[63] The present invention includes the above-described method of invivo diagnostic imaging wherein the radiolabeled inhibitor isradiolabeled with one or more radioisotope selected from ³H, ¹¹C, ¹⁴C,¹⁸F, ³²P, ³⁵S, ¹²³I, ¹²⁵I, ¹³¹I.

[64] The present invention includes the above-described method of invivo diagnostic imaging wherein wherein the inhibitor of beta-amyloidproduction is a compound selected from any compound disclosed in orwithin the scope of compounds disclosed in a reference selected from:

-   -   (1) United States patent U.S. Pat. No. 5,703,129;    -   (2) PCT application WO98/22441 (or its priority U.S. Ser. No.        08/755,444);    -   (3) PCT application WO98/22433 (or its priority U.S. Ser. No.        08/807,538);    -   (4) PCT application WO98/22430 (or its priority U.S. Ser. No.        08/754,895);    -   (5) PCT application WO98/22493 (or its priority U.S. Ser. No.        08/755,334);    -   (6) PCT application WO98/22494 (or its priorities U.S. Ser. Nos.        08/808,528, 08/807,528 or 08/807,427);    -   (7) PCT application WO98/28268 (or its priority U.S. Ser. No.        08/780,025);    -   (8) PCT application WO98/38177;    -   (9) PCT application WO95/09838;    -   (10) PCT application WO99/67221;    -   (11) PCT application WO99/67220;    -   (12) PCT application WO99/67219;    -   (13) PCT application WO95/66934;    -   (14) PCT application WO00/24392;    -   (15) Ghosh et al., JACS (2000) 122:3522-2523;    -   (16) commonly assigned attorney docket number DM-6987, U.S. Ser.        No. 09/370,089, filed Aug. 6, 1999;    -   (17) commonly assigned attorney docket number commonly assigned        attorney docket number DM-6987-A, U.S. Ser. No. 09/506,630,        filed Feb. 17, 2000;    -   (18) commonly assigned attorney docket number DM-7076, U.S. Ser.        No. 09/469,939, filed Dec. 24, 1999;    -   (19) commonly assigned attorney docket number PH-7088, filed        Feb. 17, 2000;    -   (20) commonly assigned attorney docket number PH-7131, U.S. Ser.        No. 60/158,565, filed Oct. 8, 1999;    -   (21) commonly assigned attorney docket number PH-7177, U.S. Ser.        No. 60/183,186, filed Feb. 17, 2000;    -   (22) commonly assigned attorney docket number PH-7130-P1, filed        Mar. 28, 2000;    -   (23) commonly assigned attorney docket number PH-7086-P1, filed        Mar. 31, 2000;    -   (24) commonly assigned attorney docket number PH-7164-P1, filed        Apr. 11, 2000;    -   (25) commonly assigned attorney docket number PH-7165-P1, filed        Apr. 3, 2000; or    -   (26) commonly assigned attorney docket number PH-7184-P1, filed        Apr. 3, 2000;        or any compound which inhibits beta-amyloid production and binds        competitively with any of the foregoing compounds in any of the        assays described in the Utility section hereof;        all of which foregoing references are hereby incorporated by        reference in their entirety.

[65] The present invention includes the above-described method of invivo diagnostic imaging wherein the inhibitor of beta-amyloid productionexhibits activity as an inhibitor in any of the above-described methods[1]-[15] above.

[66] The present invention includes the above-described method of invivo diagnostic imaging wherein the inhibitor of beta-amyloid productionbinds to a macromolecule which is capable of being identified by any ofthe above-described methods [19]-[27] above.

[67] The present invention includes the above-described method of invivo diagnostic imaging wherein the inhibitor of beta-amyloid productionbinds to a macromolecule as described above in [28]-[35] above.

[68] The present invention includes the above-described method of invivo diagnostic imaging wherein the inhibitor of beta-amyloid productionis selected from any of the above-described inhibitors in [36]-[42]above.

[69] The present invention includes the above-described method of invivo diagnostic imaging wherein the radiolabeled inhibitor ofbeta-amyloid production is a radiolabeled tagged inhibitor as describedabove [43]-[509 above.

[70] The present includes the above-described method of in vivodiagnostic imaging wherein the inhibitor of beta-amyloid production isselected from:

-   -   (1) an inhibitor of presenilin-1;    -   (2) an inhibitor of presenilin-2;    -   (3) an inhibitor of β secretase;    -   (4) an inhibitor of α secretase;    -   (5) an inhibitor of γ secretase; or    -   (6) an inhibitor of BACE/memapsin 2.

[71] The present invention also provides pharmaceutical compositionssuitable for in vivo diagnostic imaging comprising a radiolabeledinhibitor of beta-amyloid production. Included in the present inventionare the above-described radiolabeled inhibitors of beta-amyloidproduction, wherein the radiolabel is selected from the group ³H, ¹¹C,¹⁴C, ¹⁸F, ³²P, ³⁵S, ¹²³I, ¹²⁵I, and ¹³¹I. Included in the presentinvention is a radiopharmaceutical composition comprising aradiopharmaceutically acceptable carrier and a radiolabeled inhibitor ofbeta-amyloid production. Included in the present invention is a methodof determining levels of proteins involved in beta-amyloid production ina mammal comprising administering to the mammal a radiopharmaceuticalcomposition comprising a radiolabeled inhibitor of beta-amyloidproduction, and imaging said mammal. Included in the present inventionis a method of diagnosing a disorder associated with beta-amyloidproduction in a mammal comprising administering to the mammal aradiopharmaceutical composition comprising a radiolabeled inhibitor ofbeta-amyloid production, and imaging said mammal.

[72] The present invention includes the above-described pharmaceuticalcompositions wherein the composition is used in the diagnosis of aneurological disease which involves APP processing or elevated levels ofbeta-amyloid, or both.

[73] The present invention includes the above-described pharmaceuticalcompositions wherein the composition is used in the diagnosis ofAlzheimer's disease.

[74] The present invention includes the above-described pharmaceuticalcompositions wherein the radiolabeled inhibitor is suitable for imagingof the brain of the subject.

[75] The present invention includes the above-described pharmaceuticalcompositions wherein the radiolabeled inhibitor is radiolabeled with oneor more radioisotope selected from ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ¹²³I,¹²⁵I, ¹³¹I.

[76] The present invention includes the above-described pharmaceuticalcompositions wherein the inhibitor of beta-amyloid production is acompound selected from any compound disclosed in or within the scope ofcompounds disclosed in a reference selected from:

-   -   (1) U.S. Pat. No. 5,703,129;    -   (2) PCT application WO98/22441 (or its priority U.S. Ser. No.        08/755,444);    -   (3) PCT application WO98/22433 (or its priority U.S. Ser. No.        08/807,538);    -   (4) PCT application WO98/22430 (or its priority U.S. Ser. No.        08/754,895);    -   (5) PCT application WO98/22493 (or its priority U.S. Ser. No.        08/755,334);    -   (6) PCT application WO98/22494 (or its priorities U.S. Ser. Nos.        08/808,528, 08/807,528 or 08/807,427);    -   (7) PCT application WO98/28268 (or its priority U.S. Ser. No.        08/780,025);    -   (8) PCT application WO98/38177;    -   (9) PCT application WO95/09838;    -   (10) PCT application WO99/67221;    -   (11) PCT application WO99/67220;    -   (12) PCT application WO99/67219;    -   (13) PCT application WO95/66934;    -   (14) PCT application WO00/24392;    -   (15) Ghosh et al., JACS (2000) 122:3522-2523;    -   (16) commonly assigned attorney docket number DM-6987, U.S. Ser.        No. 09/370,089, filed Aug. 6, 1999;    -   (17) commonly assigned attorney docket number commonly assigned        attorney docket number DM-6987-A, U.S. Ser. No. 09/506,630,        filed Feb. 17, 2000;    -   (18) commonly assigned attorney docket number DM-7076, U.S. Ser.        No. 09/469,939, filed Dec. 24, 1999;    -   (19) commonly assigned attorney docket number PH-7088, filed        Feb. 17, 2000;    -   (20) commonly assigned attorney docket number PH-7131, U.S. Ser.        No. 60/158,565, filed Oct. 8, 1999;    -   (21) commonly assigned attorney docket number PH-7177, U.S. Ser.        No. 60/183,186, filed Feb. 17, 2000;    -   (22) commonly assigned attorney docket number PH-7130-P1, filed        Mar. 28, 2000;    -   (23) commonly assigned attorney docket number PH-7086-P1, filed        Mar. 31, 2000;    -   (24) commonly assigned attorney docket number PH-7164-P1, filed        Apr. 11, 2000;    -   (25) commonly assigned attorney docket number PH-7165-P1, filed        Apr. 3, 2000; or    -   (26) commonly assigned attorney docket number PH-7184-P1, filed        Apr. 3, 2000;        or any compound which inhibits beta-amyloid production and binds        competitively with any of the foregoing compounds in any of the        assays described in the Utility section hereof;        all of which foregoing references are hereby incorporated by        reference in their entirety.

[77] The present includes the above-described pharmaceutical compositionwherein the inhibitor of beta-amyloid production is selected from:

-   -   (1) an inhibitor of presenilin-1;    -   (2) an inhibitor of presenilin-2;    -   (3) an inhibitor of β secretase;    -   (4) an inhibitor of α secretase;    -   (5) an inhibitor of γ secretase; or    -   (6) an inhibitor of BACE/memapsin 2.

Definitions

As used herein, the term “Aβ” denotes the protein designated Aβ,β-amyloid peptide, and sometimes β/A4, in the art. Aβ is anapproximately 4.2 kilodalton (kD) protein of about 39 to 43 amino acidsfound in amyloid plaques, the walls of meningeal and parenchymalarterioles, small arteries, capillaries, and sometimes, venules. Theisolation and sequence data for the first 28 amino acids are describedin U.S. Pat. No 4,666,829. The 43 amino acid sequence is: 1 Asp Ala GluPhe Arg His Asp Ser Gly Tyr 11 Glu Val His His Gln Lys Leu Val Phe Phe21 Ala Glu Asp Val Gly Ser Asn Lys Gly Ala 31 Ile Ile Gly Leu Met ValGly Gly Val Val 41 Ile Ala Thr.However, a skilled artisan knows that fragments generated by enzymaticdegradation can result in loss of amino acids 1-10 and/or amino acids39-43. Thus, amino acid sequence 1-43 represents the maximum sequence ofamino acids for Aβ peptide.

The term “APP”, as used herein, refers to the protein known in the artas β amyloid precursor protein. This protein is the precursor for Aβ andthrough the activity of “secretase” enzymes, as used herein, it isprocessed into Aβ. Differing secretase enzymes, known in the art, havebeen designated β secretase, generating the N-terminus of Aβ, αsecretase cleaving around the 16/17 peptide bond in Aβ, and “γsecretases”, as used herein, generating C-terminal Aβ fragments endingat position 38, 39, 40, 41, 42, and 43 or generating C-terminal extendedprecursors which are subsequently truncated to the above polypeptides.

The compounds herein described may have asymmetric centers. Compounds ofthe present invention containing an asymmetrically substituted atom maybe isolated in optically active or racemic forms. It is well known inthe art how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.Many geometric isomers of olefins, C═N double bonds, and the like canalso be present in the compounds described herein, and all such stableisomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. All chiral, diastereomeric, racemic forms and allgeometric isomeric forms of a structure are intended, unless thespecific stereochemistry or isomeric form is specifically indicated.

The term “substituted,” as used herein, means that any one or morehydrogens on the designated atom is replaced with a selection from theindicated group, provided that the designated atom's normal valency isnot exceeded, and that the substitution results in a stable compound.When a substituent is keto (i.e., ═O), then 2 hydrogens on the atom arereplaced.

When any variable (e.g., R^(5b)) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with 0-2 R^(5b), thensaid group may optionally be substituted with up to two R^(5b) groupsand R^(5b) at each occurrence is selected independently from thedefinition of R^(5b). Also, combinations of substituents and/orvariables are permissible only if such combinations result in stablecompounds.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom on thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchsubstituent. Combinations of substituents and/or variables arepermissible only if such combinations result in stable compounds.

As used herein, “alkyl” or “alkylene” is intended to include bothbranched and straight-chain saturated aliphatic hydrocarbon groupshaving the specified number of. carbon atoms; for example, “C₁-C₆ alkyl”denotes alkyl having 1 to 6 carbon atoms. Examples of alkyl include, butare not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,sec-butyl, t-butyl, pentyl, and hexyl. Preferred “alkyl” group, unlessotherwise specified, is “C₁-C₄ alkyl”.

As used herein, “alkenyl” or “alkenylene” is intended to includehydrocarbon chains of either a straight or branched configuration andone or more unsaturated carbon-carbon bonds which may occur in anystable point along the chain. Examples of “C₂-C₆ alkenyl” include, butare not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl,2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 2-pentenyl, 3-pentenyl,hexenyl, and the like.

As used herein, “alkynyl” or “alkynylene” is intended to includehydrocarbon chains of either a straight or branched configuration andone or more carbon-carbon triple bonds which may occur in any stablepoint along the chain, such as ethynyl, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 3-butynyl, and the like.

“Alkoxy” or “alkyloxy” represents an alkyl group as defined above withthe indicated number of carbon atoms attached through an oxygen bridge.Examples of alkoxy include, but are not limited to, methoxy, ethoxy,n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, ands-pentoxy. Preferred alkoxy groups are methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, s-butoxy, t-butoxy. Similarly, “alkylthio” or“thioalkoxy” is represents an alkyl group as defined above with theindicated number of carbon atoms attached through a sulphur bridge.

“Halo” or “halogen” as used herein refers to fluoro, chloro, bromo, andiodo. Unless otherwise specified, preferred halo is fluoro and chloro.“Counterion” is used to represent a small, negatively charged speciessuch as chloride, bromide, hydroxide, acetate, sulfate, and the like.

Haloalkyl” is intended to include both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms, substituted with 1 or more halogen (for example—C_(v)F_(w) where v=1 to 3 and w=1 to (2v+1)). Examples of haloalkylinclude, but are not limited to, trifluoromethyl, trichloromethyl,pentafluoroethyl, pentachloroethyl, 2,2,2-trifluoroethyl,2,2-difluoroethyl, heptafluoropropyl, and heptachloropropyl.“Haloalkoxy” is intended to mean a haloalkyl group as defined above withthe indicated number of carbon atoms attached through an oxygen bridge;for example trifluoromethoxy, pentafluoroethoxy, 2,2,2-trifluoroethoxy,and the like. “Halothioalkoxy” is intended to mean a haloalkyl group asdefined above with the indicated number of carbon atoms attached througha sulphur bridge.

“Cycloalkyl” is intended to include saturated ring groups, having thespecified number of carbon atoms. For example, “C₃-C₆ cycloalkyl”denotes such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

As used herein, “carbocycle” is intended to mean any stable 3- to7-membered monocyclic or bicyclic or 7- to 13-membered bicyclic ortricyclic, any of which may be saturated, partially unsaturated, oraromatic. Examples of such carbocycles include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,adamantyl, cyclooctyl, [3.3.0]bicyclooctane, [4.3.0]bicyclononane,[4.4.0]bicyclodecane (decalin), [2.2.2]bicyclooctane, fluorenyl, phenyl,naphthyl, indanyl, adamantyl, or tetrahydronaphthyl (tetralin).Preferred “carbocycle” are cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

As used herein, the term “heterocycle” or “heterocyclic ring” isintended to mean a stable 5- to 7-membered monocyclic or bicyclic or 7-to 14-membered bicyclic heterocyclic ring which is saturated partiallyunsaturated or unsaturated (aromatic), and which consists of carbonatoms and 1, 2, 3 or 4 heteroatoms, preferably 1, 2, or 3 heteroatoms,independently selected from the group consisting of N, O and S andincluding any bicyclic group in which any of the above-definedheterocyclic rings is fused to a benzene ring. The nitrogen and sulfurheteroatoms may optionally be oxidized. The heterocyclic ring may beattached to its pendant group at any heteroatom or carbon atom whichresults in a stable structure. The heterocyclic rings described hereinmay be substituted on carbon or on a nitrogen atom if the resultingcompound is stable. If specifically noted, a nitrogen in the heterocyclemay optionally be quaternized. It is preferred that when the totalnumber of S and O atoms in the heterocycle exceeds 1, then theseheteroatoms are not adjacent to one another. It is preferred that thetotal number of S and O atoms in the heterocycle is not more than 1.

Examples of heterocycles include, but are not limited to, 1H-indazole,2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl,4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl, 6H-1,2,5-thiadiazinyl,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl,carbazolyl, 4aH-carbazolyl, b-carbolinyl, chromanyl, chromenyl,cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinylperimidinyl,phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl,phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl,purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl,pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl,quinolinyi, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl,triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl,1,3,4-triazolyl, xanthenyl. Preferred 5 to 10 membered heterocyclesinclude, but are not limited to, pyridinyl, pyrimidinyl, triazinyl,furanyl, thienyl, thiazolyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl,isoxazolyl, tetrazolyl, benzofuranyl, benzothiofuranyl, indolyl,benzimidazolyl, 1H-indazolyl, oxazolidinyl, isoxazolidinyl,benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, quinolinyl,and isoquinolinyl. Preferred 5 to 6 membered heterocycles include, butare not limited to, pyridinyl, pyrimidinyl, triazinyl, furanyl, thienyl,thiazolyl, pyrrolyl, piperazinyl, piperidinyl, pyrazolyl, imidazolyl,oxazolyl, isoxazolyl, tetrazolyl; more preferred 5 to 6 memberedheterocycles include, but are not limited to, pyridinyl, pyrimidinyl,triazinyl, furanyl, thienyl, thiazolyl, piperazinyl, piperidinyl,pyrazolyl, imidazolyl, and tetrazolyl. Also included are fused ring andspiro compounds containing, for example, the above heterocycles.

As used herein, the term “aryl”, “C₆-C₁₀ aryl” or aromatic residue, isintended to mean an aromatic moiety containing the specified number ofcarbon atoms; for example phenyl, pyridinyl or naphthyl. Unlessotherwise specified, “aryl” may be unsubstituted or substituted with 0to 3 groups selected from H, —OH, —OCH₃, Cl, F, Br, I, CN, —NO₂, —NH₂,—N(CH₃)H, —N(CH₃)₂, —CF₃, —OCF₃, —C(═O)CH₃, —SCH₃, —S(═O)CH₃,—S(═O)₂CH₃, —CH₃, —CH₂CH₃, —CO₂H, and —CO₂CH₃.

The phrase “additional lactam carbons”, as used herein, is intended todenote the number of optional carbon atoms in the lactam ring B ofFormula (I). Formula (I“):

represents the lactam ring B of Formula (I). Additional lactam carbonsare carbons in lactam ring B other than the carbons numbered 2 and 3 inthe backbone of the formula. The additional lactam carbons may beoptionally replaced by a heteroatom selected from oxygen, nitrogen andsulfur. Lactam ring B contains 1, 2, 3, 4, 5, 6 or 7 optional carbons,wherein one optional carbon may optionally be replaced by a heteroatom,such that the total number of members of lactam ring B, including atomsnumbered 1, 2 and 3 in the backbone, does not exceed 10. It is preferredthat the total number of atoms of lactam ring B is 6, 7 or 8; it is morepreferred that the total number of atoms of lactam ring B is seven.Examples of lactam ring B include:

but are not intended to limit the invention. Preferred examples oflactam ring B are B1, B2, B5, B6, B8, B9, B13, and B16; more preferredexamples of lactam ring B are B1, B6, B8, B9, and B13. Even morepreferred examples of lactam ring B are B1 and B6. Preferred examples ofsubstituent R¹⁰ or R¹¹ on lactam B are methyl, ethyl, phenyl,4-fluorophenyl, 4-chlorophenyl, 4-trifluorophenyl,(4-fluorophenyl)methyl, (4-chlorophenyl)methyl, and(4-trifluorophenyl)methyl.

As used herein, “macromolecule” or “complex of macromolecules”, isintended to mean a cellular component involved directly or indirectly inAPP processing and the production of Aβ peptide. By indirectly, itseffect on APP processing may be mediated by intervening molecules. Anexample of a “macromolecule” or “complex of macromolecules” ispresenilin 1 or endogenous cleavage N terminal or C terminal fragmentsof presenilin 1. Additional examples of a “macromolecule” or “complex ofmacromolecules” is presenilin 2, a homolog of presenilin 1 or a homologof presenilin 2.

It is envisaged that the scope of “macromolecule” or “complex ofmacromolecules” involved in APP processing can be found in a widevariety of sources. Sources of a “macromolecule” or “complex ofmacromolecules” are considered to be materials suspected or known tocontain a macromolecule involved in APP processing. Examples of amaterial suspected or known to contain a macromolecule involved in APPprocessing include, but are not limited to, purified proteins;suspensions of proteins; cells, tissues or organs, derived fromprokaryotes or eucaryotes; and macromolecules derived from recombinantexpression systems. Examples of cells include, but are not limited to,HEK293 cells, IMR 32 cells, RAJI cells, CHO cells, U-937 cells, andTHP-1 cells; preferably HEK293 or TNP-1 cells. Examples of tissues ororgans include, but are not limited to, spleen, brain, and testes.Examples of prokaryotes include, but are not limited to, bacteria, morepreferably E. coli. Examples of eucaryotes include, but are not limitedto, mouse, rat, guinea pig, bovine, porcine, monkey, human, andnematodes ( preferably C. elegans). An example of a suspension ofprotein includes, but is not limited to, lipid systems. An example of amacromolecule derived from recombinant expression systems includes, butis not limited to, C. elegans knockout of Sel-12 and reintroduction ofPS-1. (See Levitan, D. and Greenwald, I., Nature, 377, pp351-354, 1995.)

It is understood that one skilled in the art can readily determine thescope of the term “binding site” and “specific binding site” as usedherein. For further guidance, the tagged compounds of the presentinvention, for example (I-7T), (I-11T), and (I-43T), bind to a specificsite on one or more macromolecules involved directly or indirectly inAPP processing and the production of Aβ peptide, and thus effect adecrease in the production of Aβ peptide. One skilled in the art canreadily determine whether other compounds, which are inhibitors ofbeta-amyloid production, bind to a same site as the tagged compounds ofthe present invention by using the assays disclosed herein. However, itis understood that within the scope of the present disclosure the phrase“is identified by a compound of Formula (I-#)” or “is a specific bindingsite for a compound of Formula (I-#)” refers to defining the physicalsite on the macromolecule wherein a compound of Formula (I-#) binds toand not to a molecular reaction of binding. Thus, the phrase “a specificbinding site for a compound of Formula (I-#)” does not require thecompound of Formula (I-#) to be present.

As used herein, “potential inhibitor of beta-amyloid production” isintended to mean any compound which is being screened for activity toinhibit the production of beta-amyloid peptide, or the proteolyticactivity leading to the production of beta-amyloid peptide, using theassay of the invention described herein. It is understood that a“potential inhibitor of beta-amyloid production”, which is active in theassay of the invention for inhibiting the production of beta-amyloidpeptide, can subsequently be used in the assay of the invention as a“beta-amyloid peptide inhibitor”, as defined below, once the compoundhas been tagged. It is also understood that a “potential inhibitor ofbeta-amyloid production”, which is active in the assay of the inventionfor inhibiting the production of beta-amyloid peptide, can subsequentlybe used in pharmaceutical compositions for the treatment of degenerativeneurological disorders involving beta-amyloid production, preferably forthe treatment of Alzheimer's disease.

As used herein, “beta-amyloid peptide inhibitor” or “inhibitor ofbeta-amyloid production” is intended to mean any compound which inhibitsthe production of beta-amyloid peptide, or the proteolytic activityleading to the production of beta-amyloid peptide. Examples of abeta-amyloid peptide inhibitor include, but are not limited to, thescope of compounds of Formula (I), examples of which are disclosedherein. However, it is contemplated for use in the invention thatcompounds beyond the scope of compounds of Formula (I) may be used inthe invention. Additional examples of a beta-amyloid peptide inhibitor,contemplated by the invention, include, but are not limited to,5-amino-6-cyclohexyl-4-hydroxy-hexanamide derivatives disclosed inUnited States patent U.S. Pat. No. 5,703,129, issued Dec. 30, 1997;N-aryl amino acid esters and N-heteroaryl amino acid esters disclosed inPCT application WO98/22441 (published May 28, 1998; priority U.S. Ser.No. 08/755,444); N-arylacetyl amino acid amides, N-heteroarylacetylamino acid amides, and N-alkylacetyl amino acid amides disclosed in PCTapplication WO98/22433 (published May 28, 1998; priority U.S. Ser. No.08/807,538); N-arylacetyl amino acid esters N-heteroarylacetyl aminoacid esters, and N-alkylacetyl amino acid esters disclosed in PCTapplication WO98/22430 (published May 28, 1998; priority U.S. Ser. No.08/754,895); N-aryl amino acid derivatives and N-heteroaryl amino acidderivatives disclosed in PCT application WO98/22493 (published May 28,1998; priority U.S. Ser. No. 08/755,334); amino acid derivativesdisclosed in PCT application WO98/22494 (published May 28, 1998;priority U.S. Ser. Nos. 08/808,528, 08/807,528, 08/807,427); cycloalkyl,lactam, lactone and related compounds disclosed in PCT applicationWO98/28268 (published Jul. 2, 1998, priority U.S. Ser. No. 08/780,025);all references of which are hereby incorporated by reference in theirentirety.

As used herein, “tagged inhibitor of beta-amyloid production”, isintended to mean “beta-amyloid peptide inhibitor” compounds which aretagged. By “tagged” or “tagged inhibitor of beta-amyloid production” or“tagged compound”, it is meant that the subject beta-amyloid peptideinhibitor compounds contain a tag which is suitable for detection in anassay system or upon administration to a mammal. Suitable tags are knownto those skilled in the art and include, for example, radioisotopes,fluoroscent groups, biotin (in conjunction with streptavidincomplexation), and photoaffinity groups. As used herein, “radiolabeledcompound” or “radiolabeled inhibitor” refers to a tagged inhibitor ofbeta-amyloid production wherein the tag is a radioisotope.

For purposes of in vivo diagnostic imaging, by “radiolabeled” it ismeant that the subject inhibitors of beta-amyloid production contain aradioisotope which is suitable for administration to a mammalianpatient. Preferred radioisotopes for in vivo diagnostic imaging bypositron emission tomography (PET) are ¹¹C, ¹⁸F, ¹²³I, and ¹²⁵I.

Suitable radioisotopes are known to those skilled in the art andinclude, for example, isotopes of halogens (such as chlorine, fluorine,bromine and iodine), and metals including technetium and indium.Preferred radioisotopes include ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ¹²³I, ¹²⁵I,¹³¹I. Radiolabeled compounds of the invention may be prepared usingstandard radiolabeling procedures well known to those skilled in theart. Suitable synthesis methodology is described in detail below. Asdiscussed below, the beta-amyloid peptide inhibitor compounds of theinvention may be radiolabeled either directly (that is, by incorporatingthe radiolabel directly into the compounds) or indirectly (that is, byincorporating the radiolabel into the compounds through a chelatingagent, where the chelating agent has been incorporated into thecompounds). Also, the radiolabeling may be isotopic or nonisotopic. Withisotopic radiolabeling, one group already present in the compounds ofthe invention described above is substituted with (exchanged for) theradioisotope. With nonisotopic radiolabeling, the radioisotope is addedto the compounds without substituting with (exchanging for) an alreadyexisting group. Direct and indirect radiolabeled compounds, as well asisotopic and nonisotopic radiolabeled compounds are included within thephrase “radiolabeled compounds” as used in connection with the presentinvention. Such radiolabeling should also be reasonably stable, bothchemically and metabolically, applying recognized standards in the art.Also, although the compounds of the invention may be labeled in avariety of fashions with a variety of different radioisotopes, as thoseskilled in the art will recognize, such radiolabeling should be carriedout in a manner such that the high binding affinity and specificity ofthe unlabeled or untagged inhibitor of beta-amyloid production compoundsof the invention to the macromolecule involved in APP processing is notsignificantly affected. By not significantly affected, it is meant thatthe binding affinity and specificity is not affected more than about 3log units, preferably not more than about 2 log units, more preferablynot more than about 1 log unit, even more preferably not more than about500%, and still even more preferably not more than about 250%, and mostpreferably the binding affinity and specificity is not affected at all.

Examples of a tagged inhibitor of beta-amyloid production include, butare not limited to, the scope of compounds of Formula (I), examples ofwhich are disclosed herein. However, it is contemplated that taggedcompounds beyond the scope of compounds of Formula (I) may be used inthe invention. Additional examples of a tagged inhibitor of beta-amyloidproduction, contemplated by the invention, include, but are not limitedto, beta-amyloid peptide inhibitors disclosed in U.S. Pat. No.5,703,129, issued Dec. 30, 1997; WO98/22441 (published May 28, 1998);WO98/22433 (published May 28, 1998); WO98/22430 (published May 28,1998); WO98/22493 (published May 28, 1998); WO98/22494 (published May28, 1998); and WO98/28268 (published Jul. 2, 1998), which inhibitors canbe tagged for use in the invention. Preferred examples of a taggedinhibitor of beta-amyloid production are compounds of Formula (I) andcompounds of WO98/28268 (published Jul. 2, 1998) which can be tagged.More preferred are compounds of Formula (I).

For radiolabeled compounds, the label may appear at any position on thebeta-amyloid peptide inhibitor. Preferred radiolabeled compounds of theinvention are beta-amyloid peptide inhibitor radiolabeled with tritium.More preferred radiolabeled compounds of the invention are radiolabeledcompounds wherein the radiolabel is located on R³ of Formula (I).

As used herein, when the tagged inhibitor of beta-amyloid production istagged with a photoaffinity group or photoaffinity labeled, the term“photoaffinity group” or “photoaffinity labeled” refers to a substituenton the inhibitor which can be activated by photolysis at an appropriatewavelength to undergo a crosslinking photochemical reaction with themacromolecule to which it is associated. An example of a “photoaffinitygroup” is a benzophenone substituent.

In the present invention it has also been discovered that theradiolabeled compounds above are useful as inhibitors of beta-amyloidpeptide production and thus the radiolabeled compounds of the inventionmay also be employed for therapeutic purposes, in addition to thediagnostic usage described above.

As used herein, “inhibitory concentration” is intended to mean theconcentration at which the “potential inhibitor of beta-amyloidproduction” compound screened in the assay of the invention inhibits ameasurable percentage of beta-amyloid peptide production. Examples of“inhibitory concentration” values range from IC₅₀ to IC₉₀, and arepreferably, IC₅₀, IC₆₀, IC₇₀, IC₈₀, or IC₉₀, which represent 50%, 60%,70%, 80% and 90% reduction in beta-amyloid peptide production,respectively. More preferably, the “inhibitory concentration” ismeasured as the IC₅₀ value. It is understood that an designation forIC₅₀ is the half maximal inhibitory concentration.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,benzenesulfonic, toluenesulfonic, methanesulfonic, ethane disulfonic,oxalic, isethionic, and the like.

The pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, the disclosure of which is hereby incorporated byreference.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

Synthesis

The compounds of the present invention can be prepared in a number ofways well known to one skilled in the art of organic synthesis. Thecompounds of the present invention can be synthesized using the methodsdescribed below, together with synthetic methods known in the art ofsynthetic organic chemistry, or variations thereon as appreciated bythose skilled in the art. Preferred methods include, but are not limitedto, those described below. All references cited herein are herebyincorporated in their entirety herein by reference.

The novel compounds of this invention may be prepared using thereactions and techniques described in this section. The reactions areperformed in solvents appropriate to the reagents and materials employedand are suitable for the transformations being effected. Also, in thedescription of the synthetic methods described below, it is to beunderstood that all proposed reaction conditions, including choice ofsolvent, reaction atmosphere, reaction temperature, duration of theexperiment and workup procedures, are chosen to be the conditionsstandard for that reaction, which should be readily recognized by oneskilled in the art. It is understood by one skilled in the art oforganic synthesis that the functionality present on various portions ofthe molecule must be compatible with the reagents and reactionsproposed. Such restrictions to the substituents which are compatiblewith the reaction conditions will be readily apparent to one skilled inthe art and alternate methods must then be used.

Methods for the synthesis of succinylamino lactams are known in the artand are disclosed in a number of references including PCT publicationnumber WO 96/29313, which is hereby incorporated by reference.

Disubstituted succinate derivatives can be prepared by a number of knownprocedures. The procedure of Evans (D. A. Evans et al, Org. Synth. 86,p83 (1990)) is outlined in Scheme 1 where acylation of an oxazolidinonewith an acylating agent such as an acid chloride provides structures 1.Alkylation to form 2 followed by cleavage of the chiral auxiliary andsubsequent alkylation of the dianion of the carboxylic acid 3 provides avariety of disubstituted succinates which can be separated andincorporated into structures of Formula (I) by those skilled in the art.Additional examples are found in P. Becket, M. J. Crimmin, M. H. Davis,Z. Spavold, Synlett, (1993), 137-138, incorporated herein by reference.

Diastereomerically pure succinate derivatives can be accessed using thechemistry outlined below, adapted from P. Becket, M. J. Crimmin, M. H.Davis, Z. Spavold, Synlett, (1993), 137-138 incorporated herein byreference. This reference provides the synthesis below to obtaincompound 9. Compound 11 is used as an intermediate and is prepared from9 by hydrogenation of the allyl group followed by coupling of9-fluorenemethanol under standard conditions using DCC and DMAP inCH₂Cl₂. Deprotection of the tert-butyl ester is accomplished bytreatment with 50% trifluoroacetic acid.

Additional methods useful for the preparation of succinate derivativesare known by those skilled in the art. Such references include, McClureand Axt, Bioorganic & Medicinal Chemistry Letters, 8 (1998) 143-146;Jacobson and Reddy, Tetrahedron Letters, Vol 37, No. 46, 8263-8266(1996); Pratt et al., SYNLETT, May 1998, p. 531.

A variety of compounds of Formula (I) can be prepared by methodsdescribed in Scheme 4. The protected α-amine 3 of theα-amino-ε-caprolactam can be prepared by methods well known in theliterature for amino protecting groups as discussed in Theodora W.Greene's book “Protective Groups in Organic Synthesis”, such as N-Bocusing di-t-butyldicarbonate in an appropriate solvent like DMSO. Asulfur atom can be introduced into the ring providingL-α-amino-β-thio-ε-caprolactam according to the procedure in S. A. Ahmedet al, FEBS Letters, (1984), vol. 174, pages 76-9 (Scheme 3). Oneskilled in the art can extend this methodology to the synthesis ofβ-amino and oxygen containing rings by analogy. The sulfur-containingmolecules can also be oxidized to the sulfoxide and sulfone by methodsknown to one skilled in the art.

The lactam nitrogen of compound 13 can be alkylated by generating theanion with bases such as LDA, lithium bis(trimethylsilyl)amide or sodiumhydride in solvents like THF, with or without cosolvents such as DMPU orHMPA and reacting this with a variety of groups containing leavinggroups (X″) like bromide, iodide, mesylate or tosylate. Alkylatingagents such as a-bromo amides, ketones and acids can be prepared by anumber of literature methods including halogenation of amino acids bydiazotization or are commercially available. Other suitable alkylatingagents such as alkyl, allylic and benzylic halides can be formed form avariety of precursors such as free-radical addition of halides oractivation of alcohols, and other chemistries known to those skilled inthe art. For discussion of these types of reactions, see Carey, F. A.and Sundberg, R. J., Advanced Organic Chemistry, Part A, New York:Plenum Press, 1990, pages 304-305, 342-347, 695-698.

The N-Boc protecting group can be removed by any number of methods wellknown in the literature like TFA in methylene chloride to give thecompound 15. The amine 15 can be coupled to an appropriately substitutedcarboxylic acid or acid chloride by methods well described in theliterature for making amide bonds, like TBTU in DMF with a base like NMMto give the elaborated compound 16. Compounds 16 can be alkylated usingstandard bases like LDA, NaH, or NaHMDS to deprotonate the amidefollowed by addition of an alkylating agent with an appropriate leavinggroup like halide, mesylate, or triflate in an appropriate solvent toprovide compounds 17 with an R⁶ substituent. The t-butyl ester is thenremoved by treatment with TFA in methylene chloride to give thecarboxylic acid 17.

The final compounds 18 were prepared by treating the activatedcarboxylic acid of 17 with an appropriately substituted amine. Forinstance, activation of the carboxylic acid with HATU(O-(7-azabenzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumhexafluorophosphate) or PyBOP(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate) or other coupling agents known to those skilled inthe art allows condensation with ammonia to form primary amides.Similarly, condensation of the activated acid with hydroxylaminehydrochloride provides the hydroxamic acid, or reaction with a primaryor secondary amine provides the substituted amine derivative.

Activation of the acid with PyBrOP (bromo-tris-pyrrolidino-phosphoniumhexafluorophosphate) followed by addition of an alcohol and4-dimethylaminopyridine allows formation of the ester directly. Foradditional acylation reactions see for example Carey, F. A. andSundberg, R. J., Advanced Organic Chemistry, Part A, New York: PlenumPress, 1990, pages 475-479.

Additional Examples of compounds of Formula (I) can be prepared as shownin Scheme 5. A suitable resin for solid phase synthesis such as Fmoc(Fluorenylmethylcarbonyl)-protected hydroxylamine bound to polystyrenebeads can be purchased from Novabiochem, Inc. Deprotection of the Fmocgroup under standard conditions using 20% piperidine in DMF providestrityl-linked hydroxylamine resin. Coupling of afluorenylmethyl-protected succinic acid derivative such as 20 with acoupling agent such as HATU in a suitable solvent like DMF orN-methylpyrrolidinone provides the support-bound hydroxamate 21. TheFluorenylmethyl ester can be removed using 20% piperidine in DMF toprovide the free carboxylic acid which can be coupled to amines like thecaprolactam 22 (which is available using chemistry outlined in Scheme 4)using PyBOP (benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate) and a suitable base like DIEA in DMF or NMP. Thesupport-bound intermediate 23 can then be elaborated to biarylstructures of the type 24 using typical Suzuki coupling conditionsemploying a catalyst such as Palladium complexes liketetrakis(triphenylphosphine)-palladium with 2M aqueous sodium carbonateas a base in a suitable solvent like THF or DME and an excess of aboronic acid. The final compounds are liberated from the supportemploying dilute (5%) trifluoroacetic acid in CH₂CL₂ and purified byconventional chromatography.

General procedure for solid-phase synthesis according to Scheme 5.

Resin 20 of Scheme 5: Fmoc-protected resin 19 (2.0 g, 0.78 mmol/g, 1.56mmol) is purchased from Novabiochem and swelled in 20 ml of CH₂Cl₂ for 1hour. The CH₂Cl₂ is removed and the resin is then treated with 25% v/vpiperidine in DMF (8 mL) and allowed to shake slowly for 16 h. Thesolvent was removed by filtration and the resin was shaken with anadditional 8 mL of 25% v/v piperidine in DMF for 2 h at rt. The solventswere removed by filtration, and the resin 20 was rinsed 3× with 20 mL ofDMF, 3× with 20 mL of methanol, and 3× with 20 mL of CH₂Cl₂ and dried invacuo.

Succinate 10 of Scheme 2: Succinate 9 is prepared according to theliterature procedure (P. Becket, M. J. Crimmin, M. H. Davis, Z. Spavold,Synlett, (1993), 137-138). Succinate 9 (17.8 g, 66 mmol) is dissolved in250 mL of ethyl acetate and placed in a Parr shaker bottle. To thesolution is added 890 mg of 5% palladium on carbon, and the bottle ispressurized to 40 psi with hydrogen gas and shaken for 2.5 h at rt. Thehydrogen is removed and the palladium catalyst is removed by filtrationthrough a pad of celite. Concentration of the ethyl acetate solutionprovides 17.5 g (98%) of succinate 10. No further purification isnecessary. MS (M-H)⁺=271.

Succinate 21 of Scheme 5: Succinate 10 (6.3 g, 23.1 mmol) is dissolvedin 125 mL of CH₂Cl₂ and 4.8 g (23.3 mmol) of dicyclohexylcarbodiimide isadded. The solution is stirred at rt for 30 min and then 4.6 g (23.4mmol) of 9-fluorenemethanol is added followed by 122 mg (1 mmol) of4-dimethylaminopyridine. After 5 h of stirring at rt, the reactionsolution was diluted with an additional 100 mL of CH₂Cl₂ and filteredthrough a pad of celite to remove precipitated dicyclohexylurea. Thesolution was then washed 3× with 50 mL of a 1N HCl solution, 3× with 50mL of a saturated sodium bicarbonate solution, and 2× with 50 mL ofbrine. The crude product was dried over MgSO₄ and soncentrated onto 15 gof silica gel. Chromatography eluting with a gradient of 2.5% to 5%ethyl acetate/hexanes provided 6.4 g (61%) of the diester as an oil. Thepurified diester (6.4 g 14.2 mmol) is then dissolved in 25 mL of CH₂Cl₂, 25 mL of trifluoroacetic acid is added, and the reaction solution isstirred at rt for 2 h. The reaction solution is directly concentrated invacuo to an oil which is then redissolved in 25 mL of toluene andreconcentrated, followed by drying in vacuo to provide 6.3 g (98%) ofthe desired succinate 9 as an oil which solidifies on standing. MS(M+Na)⁺=471, (M+2Na)⁺=439.

Caprolactam 23 of Scheme 5: Boc-caprolactam 14 (5.0 g, 21.9 mmol) isdissolved in 60 mL of THF and chilled to −78° C. To the chilled solutionis added 24 mL of a 1.0 M solution of lithium bis(trimethylsilyl)amidein THF, and the solution was brounght to 0° C. and stirred for 15 min.To the anion solution was added 6.5 g (22 mmol) of 3-iodobenzyl bromide(Aldrich) and the the solution was allowed to warm to rt and stirred for18 h. The reaction solution was diluted with 50 mL of water andextracted 3× with ethyl acetate. The combined organic layers were driedover MgSO₄ and concentrated in vacuo. The crude product was purified bychromatography eluting with a gradient of 5-20% ethyl acetate/hexanes toafford 7.0 g (72%) of the title compound as a white solid. MS(M+Na)⁺=467.

Resin 22 of Scheme 5: Resin 22 (2.0 g, 0.78 mmol/g, 1.56 mmol) wasswollen in 3 mL of DMF. In a separate flask, 1.85 g (4.68 mmol) ofsuccinate 21 was dissolved in 3 mL of DMF and 2.5 mL ofN,N-diisopropylethylamine (14 mmol) wsa added, followed by 1.81 g (4.68mmol) of HATU. The solution containing the active ester was added to theslurried resin and the reaction suspension was slowly shaken for 18 h.The resin was then washed 3× with 20 mL of DMF, 3× with 20 mL ofmethanol, and 3× with 20 mL of CH₂Cl₂. Loading of the resin wasdetermined by Fmoc quantitation to be 0.25 mmol/g, see Reddy, M. P.;Voelker, P. J. Int. J. Pept. Protein Res. 1998, 31, 345-348.

Resin 24 of Scheme 5: Resin 22 (2.0 g , 0.25 mmol/g, 0.5 mmol) wassuspended in 10 mL of 25% piperidine in DMF. The suspended resin wasshaken for 30 min at rt, and then the resin was washed 3× with 20 mL ofDMF, 3× with 20 mL of methanol, and 3× with 20 mL of CH₂Cl₂. Deprotectedresin (1.0 g, 0.25 mmol) was swollen in 2 mL of DMF. To the slurry wasadded 650 mg (1.25 mmol) of PyBOP and 217 mL (1.25 mmol) of DIEA.Separately, 443 mg (0.97 mmol) of caprolactam 23 was dissolved in 2 mLof DMF and 436 mL (2.5 mmol) of DIEA was added. The caprolactam solutionwas added to the resin slurry and the resin was mixed for 18 h at rt.The solvents were then removed and the coupling was repeated, withshaking at rt for 6 h. The resin was then washed 3× with 10 mL of DMF,3× with 10 mL of methanol, and 3× with 10 mL of CH₂Cl₂.

Products 25 of Scheme 5: A 70 mg (17.5 mmol) portion of resin 24 wassuspended in 1 mL of THF in a screw-cap vial. To the slurry was added aboronic acid (0.15 mmol), 150 mL of a 2 M solution of sodium carbonate,and 15 mg (13 mmol) of tetrakis(triphenylphosphine)palladium. The vialwas tightly closed and heated to 60° C. for 16 h using a dry heater on ashaker table. The solvents were then removed by filtration and the resinwas washed 3× with THF (2 mL), 3× with methanol (2 mL), 3× with water,and 3× with CH₂Cl₂. The resins were then placed in a glass vial andcleaved with 1 mL of 5% trifluoroacetic acid in CH₂Cl₂ for 30 min. Thesolution ws filtered off and the resin was washed with an additional 2mL of CH₂Cl₂ and the combined filtrates were evaporated to dryness toyield the crude products 25. The products were purified bychromatography eluting with 10-100% ethyl acetate in hexanes to yield13.0 to 6.0 mg (14-60%) of the final products.

Additional Examples of compounds of Formula (I) can be prepared as shownin Scheme 6. A suitable resin for solid phase synthesis such as Fmoc(Fluorenylmethylcarbonyl)-protected peptide amide linker(PAL)-derivatized polystyrene beads can be purchased from Perkin ElmerBiosystems, Inc. Deprotection of the Fmoc group under standardconditions using 20% piperidine in DMF provides the free benzylamine.Coupling of a succinic acid derivative such as 28 (which is availableusing chemistry outlined in Scheme 4) with a coupling agent such as HATUin a suitable solvent like DMF or N-methylpyrrolidinone provides thesupport-bound amide 29. The support-bound intermediate 29 can then beelaborated to biaryl structures of the type 24 using typical Suzukicoupling conditions employing a catalyst such as Palladium complexeslike tetrakis(triphenylphosphine)-palladium with 2M aqueous sodiumcarbonate as a base in a suitable solvent like THF or DME and an excessof a boronic acid. The final compounds are liberated from the supportemploying 50% trifluoroacetic acid in CH₂Cl₂ and can be purified byconventional chromatography or preparative HPLC.

General Procedure for Solid-Phase Synthesis According to Scheme 6

Resin 27 of Scheme 6: Fmoc-protected PAL resin 26 (0.80 g, 0.50 mmol/g,0.40 mmol) is purchased from Advanced Chemtech and swelled in 20 ml ofCH₂Cl₂ for 1 hour. The CH₂Cl₂ is removed and the resin is then treatedwith 25% v/v piperidine in DMF (6 mL) and allowed to shake slowly for 1h. The solvents were removed by filtration, and the resin 27 was rinsed3× with 20 mL of DMF, 3× with 20 mL of methanol, and 3× with 20 mL ofCH₂Cl₂ and dried in vacuo.

Acid 28 of Scheme 6: To a solution of 0.100 g (367 mmol) of succinate 10dissolved in 2.0 mL of dry DMF was added 0.120 mL (1.10 mmol) ofN-methylmorpholine. A second solution containing 0.139 g (0.403 mmol) ofcaprolactam 23 of Scheme 5 dissolved in 2.0 mL of DMF was then added. Tothe mixed solution was added 229 mg (0.440 mmol) of PyBop and thereaction solution was stirred for 16 h at rt. The reaction solution wasdiluted with water (20 mL) and extracted 3× with 100 mL of ethylacetate. The combined organic layers were dried with Na₂SO₄ andconcentrated under reduced pressure. The resulting oil was purified bychromatography eluting with a gradient of 5-20% ethyl acetate in hexanesto provide 0.195 g (0.360 mmol, 98%) of the tert-butyl ester of Acid 28(MS M+Na=621). The purified ester (0.195 g, 0.360 mmol) was dissolved in10 mL of 25% trifluoroacetic acid in CH₂Cl₂ and stirred for 2 h at rt.The solvents were removed under reduced pressure and the acid wasredissolved in 5 mL of toluene and reconcentrated 2× to remove residualTFA. The crude acid was found to be pure by ¹H NMR and was used inScheme 6 without further purification.

Resin 29 of Scheme 6. Resin 27 (800 mg, 0.40 mmol) was solvated in 4.0mL of dry DMF and and 0.63 mL (3.6 mmol) of diisopropylethylamine wasadded followed by a solution of Acid 28 dissolved in 4 mL of DMF. To theslurry was then added 0.465 g (1.2 mmol) of HATU and the slurry wasshaken for 26 h at rt. The solvents were removed by filtration, and theresin 29 was rinsed 3× with 20 mL of DMF, 3× with 20 mL of methanol, and3× with 20 mL of CH₂Cl₂. and dried in vacuo.

Products 30 of Scheme 6: A 75 mg (0.38 mmol/g, 28.8 μmol) portion ofresin 24 was suspended in 1 mL of THF in a screw-cap vial. To the slurrywas added a boronic acid (0.33 mmol), 150 mL of a 2 M solution of sodiumcarbonate, and 15 mg (13 mmol) of tetrakis(triphenylphosphine)palladium.The vial was tightly closed and heated to 60° C. for 16 h using a dryheater on a shaker table. The solvents were then removed by filtrationand the resin was washed 3× with THF (2 mL), 3× with methanol (2 mL), 3×with water, and 3× with CH₂Cl₂. The resins were then placed in a glassvial and cleaved with 1 mL of 5% trifluoroacetic acid in CH₂C₁₂ for 2 h.The solution was filtered off and the resin was washed with anadditional 2 mL of CH₂Cl₂ and the combined filtrates were evaporated todryness to yield the crude products 25. The products were purified bychromatography eluting with 10-100% ethyl acetate in hexanes to yield0.5 to 2.0 mg (14-60%) of the final products.

The internal phenyl ring can be exchanged for a pyridine ring usingchemistry outlined in Scheme 7. The chloromethyl pyidine 33 is preparedusing a known procedure reported in Nutaitis, Charles F.; Ledeboer, MarkW. Org. Prep. Proced. Int. (1992), 24(2), 143-6 Incorporated herein byreference. After freebasing the pyridine, alkylation with theBoc-caprolactam provides pyridine intermediate 34, which can beelaborated to the protected amide 35 with succinate 10. Substitution canthen be introduced using Suzuki methodology employing a palladium sourcesuch as tetrakis(triphenylphosphine) palladium(0) orbis(diphenylphosphinoferrocene) palladium(II) dichloride and a suitablebase such as sodium carbonate or triethylamine in a solvent such as THFor toluene containing 10% methanol. Stille chemistry is also possibleusing a suitable palladium source such astetrakis(triphenylphosphine)palladium(0) and an aryl or vinyl tinderivative in a solvent such as benzene, toluene, or xylenes. Thetert-butyl ester is then deprotected under standard acidic conditionsusing trifluoroacetic acid and the amide is formed under standardconditions to provide products 36.

General Procedure for Synthesis According to Scheme 7

The chloromethyl pyidine HCl salt 33 is prepared using a known procedurereported in Nutaitis, Charles F.; Ledeboer, Mark W. Org. Prep. Proced.Int. (1992), 24(2), 143-6.

Caprolactam 34: Pyridine HCl salt 33 (2.0 g, 8.3 mmol) is dissolved in50 mL of a saturated NaHCO₃ solution and the solution is extracted with30 mL of CH₂Cl₂ 3× followed by concentration of the organic layers toprovide the free base. Separately, 1.8 g (7.8 mmol) of caprolactam 13 isdissolved in 40 mL of dry THF and chilled to −78° C. To the solution wasadded 8.7 mL of a 1M solution of sodium bis(trimethylsilyl) amide. Thesolution was brought to 0° C. and stirred for 30 min. To the resultantanion was added a solution of 1.7 g (8.3 mmol) of pyridine 33 free basedissolved in 40 mL of THF. The resulting reaction solution was stirredat rt for 18 h and then heated to 50° C. and stirred an additional 3 h.The reaction solution was allowed to cool and then 50 mL of water wasadded and the aqueous layer was extracted 2× with 100 mL of ethylacteate. The combined organic layers were dried and concentrated underreduced pressure to provide the crude product which was purified bychromatography eluting with 20 to 100% ethyl acetate in hexanes toprovide 1.5 g (51%) of caprolactam 34 as an oil.

Amide 35: Caprolactam 34 (0.40 g, 1.0 mmol) is dissolved in 20 mL of 50%trifluoroacetic acid in CH₂Cl₂ and stirred at rt for 30 min. Thesolvents were then removed under reduced pressure and the resulting oilwas redissolved in 5 mL of toluene and reconcentrated to remove residualTFA. Separately, 0.270 g (1.0 mmol) of succinate 10 was dissolved in 5.0mL of dry DMF and 0.44 mL (4 mmol) of N-methylmorpholine was addedfollowed by 0.50 g (1.3 mmol) of HATU and the resulting solution wasstirred at rt for 30 min. The crude deprotected caprolactam from abovewas dissolved in 5.0 mL of dry DMF and added to the succinate solutionand the resulting solution was heated to 50° C. and stirred for 2 days.The solution was then diluted with 20 mL of water and extracted with 350 mL portions of ethyl acetate. The combined organic layers were driedand concentrated under reduced pressure to provide an oil which waspurified by chromatography eluting with 20 to 50% ethyl acetate inhexanes to provide 0.40 g (70%) of the Amide 35.

The compounds of Formula (I) of the present invention can also beprepared from aminolactam 42 and succinic acid derivatives 41 usingamide bond syntheses known in the art, including methods commonly usedin peptide syntheses, such as HATU, TBTU, BOP, pyBOP, EDC, CDI, DCC,hydroxysuccinimide, mixed carboxylic anhydride, and phenyl estermediated couplings, as illustrated in Scheme 9 for the synthesis ofaminolactam 43, an embodiment of the present invention.

Depending on the structure of the final product, it is appreciated bythose skilled in the art that protecting groups or precursorfunctionality convertable to the desired groups may be desireable.Protecting groups and their use in synthesis are described in Green andWuts, Protective Groups in Organic Synthesis, (Wiley 1991). The use ofprotecting groups is further illustrated in Scheme 10, in which thesuccinate half-ester 44 (Becket et al., Synlett 1993, 137-138) iscoupled to the aminobenzodiazepine 45 (Sherrill and Sugg, J. Org. Chem.1995, 60, 730-734; Bock et al., J. Med. Chem., 1993, 36, 4276-4292) togive ester 46, followed by conversion of the ester group to the primaryamide 47.

Methods for the synthesis of lactams as contemplated by the presentinvention in lactam ring B in Formula (I), including aminobenzodiazepines, are known in the art and are disclosed in a number ofreferences including PCT publication number WO 98/28268, which is herebyincorporated by reference. Additional references include Bock, et al, J.Org. Chem., 1987, 52, 3232-3239 and Sherrill et al, J. Org. Chem., 1995,60, 730-734; Walsh, D. A., Synthesis, September 1980, p.677.

EXAMPLES

Chemical abbreviations used in the Examples are defined as follows:“DMPU” for 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidone, “TBTU” forO-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate,and “BOP” for benzotriazol-1-yloxytris-(dimethylamino)phosphoniumhexafluorophosphate. It is understood that one skilled in the art candiscern compounds used in the synthesis of Examples of the invention maybe referred to by structure and number. For example, Resin 20 refers tothe resin of structure 20 in Scheme 5; succinate 9 refers to thestructure 9 found in Scheme 2 which is a succinate compound.

“HPLC” is an abbreviation used herein for high pressure liquidchromatography. Reverse-phase HPLC was carried out using a Vydac C-18column with gradient elution from 10% to 100 % buffer B in buffer A(buffer A: water containing 0.1% trifluoroacetic acid, buffer B: 10%water, 90% acetonitrile containing 0.1% trifluoroacetic acid).

Example 1 (2R,3S)N1-[(3S)-hexahydro-1-(3,3-diphenylpropyl)-2-oxo-1H-azepin-3-yl]-N4-(hydroxy)-2-(2-methylpropyl)-3-(propyl)-butanediamide

Step (1a): Di-tert-butyldicarbonate (10.2 g, 46.7 mmoles) was addedportion wise to a solution of L-(−)-α-amino-ε-caprolactam (5.0 g, 39.0mmoles) in dimethyl sulfoxide (30 mL). After 5 h at rt, the reaction waspartitioned between water (100 mL) and ethyl acetate. The combinedorganic extracts were washed successively with 1 M HCl (50 mL), brine,and dried (MgSO₄) and concentrated in vacuo. The residue wasrecrystallized in 1:1 v/v ether-hexanes, two crops yielded the desiredproduct (6.26 g, 70%) as white solid. MS (M+H−BOC)⁺=129.

Step (1b): Triphenylphosphine (3.0 g, 11.4 mmoles) and carbontetrabromide (3.75 g, 11.7 mmoles) were added successively to a cooled(0° C.) solution of 3,3-biphenyl-1-propanol (1.5 mL, 7.5 mmoles) indichloromethane (20 mL). After 1.5 hours at rt, the mixture wasconcentrated in vacuo. The residue was purified by flash chromatographyon silica gel (hexanes) to give the desired product (1.93 g, 93% yield)as a clear oil. MS (M−BrC₂H₄)⁺=167

Step (1c): A 1.0 M tetrahydrofuran solution of lithiumbis(trimethylsilyl)amide (1.3 mL) was added over 15 minutes to compoundof Step (1a) (0.29 g, 1.27 mmoles) in tetrahydrofuran (3 mL) and DMPU (2mL) at −78° C. The iodo compound prepared from compound (1b) (0.85 g,3.09 mmoles) by typical Finkelstein methodology, in tetrahydrofuran (4mL) was added and the reaction was allowed to warm to rt slowly. Thiswas stirred for 10 hours at ambient temperature, partitioned betweenwater and ethyl acetate. The combined organic extracts were washedsuccessively with water (20 mL), brine (20 mL), and dried (MgSO₄) andconcentrated in vacuo. The resulting residue was purified by silica gelcolumn (ethyl acetate:hexanes, 5:95 then ethyl acetate:hexanes, 15:85)to give the desired product (0.16 g, 30%). MS (M−Ot−Bu)⁺=349.

Step (1d): Trifluoroacetic acid (3 mL) was added to a solution ofcompound of Step (1c) (0.16 mg, 0.38 mmoles) in dichloromethane (9 mL).After 2 h at rt, the solvent was removed in vacuo. The residualtrifluoroacetic acid was removed by azeotrope with dichloromethane (50mL), toluene (50 mL), and dichloromethane (50 mL) successively to givethe desired product (0.17 g, 99%) as a yellow oil. MS (M+H)⁺=323.

Step (1e): 4-Methylmorpholine (0.6 mL, 5.46 mmoles) and TBTU (0.11 g,0.34 mmoles) were added to a solution of succinate acid (P. Becket, M.J. Crimmin, M. H. Davis, Z. Spavold, Synlett, (1993), 137-138) (0.085 g,0.31 mmoles) in N,N-dimethylformamide (3 mL). After 30 minutes at rt,the compound from step (1d) (0.17 g, 0.39 mmoles) was added to themixture. The reaction was stirred for 16 h at rt, then partitionedbetween 1 M HCl (20 mL) and ethyl acetate. The combined organic extractswere washed successively with saturated aqueous sodium bicarbonate (20mL), water (20 mL), brine (20 mL), dried (MgSO₄) and concentrated invacuo. The residue was purified by silica gel chromatography (ethylacetate:hexanes, 7:93 gradient to ethyl acetate:hexanes 25:75) to givethe desired product (120 mg, 67%) as a clear oil. MS (M+NH₄−Ot−Bu)⁺=521.

Step (1f): Trifluoroacetic acid (3 mL) was added to a solution ofcompound of Step (1e) (120 mg, 0.21 mmoles) in dichloromethane (9 mL).After 3 hours at rt, the mixture was concentrated in vacuo. The residualtrifluoroacetic acid was removed by azeotrope with toluene (1×50 mL) anddichloromethane (1×50 mL). The residue was triturated with Et₂O:Hexanes95:5, to give the desired product (75 mg, 70%) as a white solid. MS(M−H)⁻=519.

Step (1g): 4-Methylmorpholine (0.05 mL, 0.45 mmoles) and BOP (73 mg,0.17 mmoles) were added to a solution of compound of Step (1f) (60 mg,0.12 mmoles) in N,N-dimethylformamide (2 mL). Hydroxylamine (33 mg, 0.47mmoles) was added to the mixture, the reaction was stirred for 16 h atrt, was concentrated in vacuo, was acidified with trifluoroacetic acid,then purified by reverse phase HPLC on a Vydac C-18 column, to give thedesired hydroxamic acid as a white solid (45 mg, 75%). MS (M−H)⁻=534.

Example 2 (2R,3S)N1-[(3S)-hexahydro-1-(3-phenoxybenzyl)-2-oxo-1H-azepin-3-yl]-N4-(hydroxy)-2-(2-methylpropyl)-3-(propyl)-butanediamide

Step (2a): Triphenylphosphine (3.40 g, 13.0 mmoles) andcarbontetrabromide (4.20 g, 13.0 mmoles) were added successively to asolution of m-phenoxybenzyl alcohol (1.5 mL, 8.6 mmoles). After 4 h atrt the mixture was concentrated and was purified by silica gel column(hexanes, then ethyl acetate:hexanes, 5:95) to give the desired bromide(1.3 g, 57%) as a yellow oil. MS (M−Br)⁺=183.

Step (2b): A 1 M solution of lithium bis(trimethylsilyl)amide was addeddropwise to a solution of compound of Step (1a) (0.3 g, 1.31 mmoles) intetrahydrofuran (5 mL) at −78° C. After 30 minutes a solution ofcompound of Step (2a) (0.43 g, 1.63 mmoles) in tetrahydrofuran (4 mL)was added to the mixture dropwise. The reaction was allowed to come toambient temperature, stirred for 16 h, then partitioned between waterand ethyl acetate. The combined organic extracts were washedsuccessively with water (20 mL), brine (20 mL), dried (MgSO₄) andconcentrated in vacuo. The crude residue was purified by silica gelchromatography (ethyl acetate:hexanes, 5:95 then ethyl acetate:hexanes,15:85) to give the desired product (360 mg, 67%) as a clear oil. MS(M−Ot−Bu)⁺=337.

Step (2c): Trifluoroacetic acid (5 mL) was added to a solution ofcompound of Step (2b) in dichloromethane (15 mL). After 3 h at rt thesolution was concentrated in vacuo. The residual trifluoroacetic acidwas removed from residue by azeotrope with toluene (50 mL) thendichloromethane (30 mL) to yield the desired amine (390 mg, 99%) as aclear oil. MS (M+H)⁺=311.

Step (2d): Following a procedure analogous to the preparation of Step(1e), but using the compound from of Step (2c) (390 mg, 0.88 mmoles) theamide was prepared, The crude compound was purified by silica gelchromatography to give the desired product (0.38 g, 92%) as a yellowoil. MS (M−Ot−Bu)⁺=491.

Step (2e): Following a procedure analogous to the preparation of step(1f), but using the compound from Step (2d) (380 mg, 0.67 mmoles), thecarboxylic acid was prepared. The product was precipitated from ethylether with hexanes, to give the desired acid (227 mg, 66%) as a whitesolid. MS (M−H)⁻=507.

Step (2f): Following a procedure analogous to the preparation ofcompound of Step (1g), but using the compound from step (2e) (150 mg,0.29 mmoles) the title compound was prepared. The crude was purified byreverse phase HPLC on a Vydac C-18 column to give the desired product(90 mg, 58%) as a white solid. MS (M−H)⁻=522.

Example 3 (2R,3S)N1-[(3S)-hexahydro-1-(phenyl)-2-oxo-1H-azepin-3-yl]-N4-(hydroxy)-2-(2-methylpropyl)-3-(propyl)-butanediamide

Step (3a): Triethylamine (1.5 mL, 10.8 mmoles), copper (II) acetate(0.95 g, 5.2 mmoles) and phenylboric acid (1.6 g, 13.1 mmoles) wereadded successively to a solution of compound of Step (1a) (1.0 g, 4.4mmoles) in dichloromethane (20 mL). After 2.5 h at rt, more phenylboricacid (0.5 g, 4.1 mmoles) was added to the mixture. After an additional 3hours at rt more phenylboric acid (0.5 g, 4.1 mmoles) was added to themixture. After 65 h at rt, the mixture was filtered over celite. Thefiltrate was concentrated in vacuo, and the residue was purified bysilica gel chromatography (ethyl acetate:hexanes, 5:95 then 15:85) togive the desired product (250 mg, 19%). MS (M−Ot−Bu)⁺=231.

Step (3b): Following a procedure analogous to the preparation ofcompound of Step (2c), but using compound of Step (3a) (250 mg, 0.82mmoles), the amine (300 mg, 99%) was prepared as a yellow oil. MS(M+H)⁺=205.

Step (3c): Following a procedure analogous to the preparation ofcompound of Step (1e), but using compound from Step (3b) (0.3 g, 0.94mmoles), the amide was prepared. The residue was purified by silica gelchromatography (ethyl acetate:hexanes, 5:95 to 20:80 in 5% increments,500 mL each ratio) to give the desired product (210 mg, 60%) as a clearoil. MS (M+H−t−Bu)⁺=403.

Step (3d): Following a procedure analogous to the preparation ofcompound of Step (1f), but using compound from sStep (3c) (200 mg, 0.44mmoles) the acid was prepared. The crude oil was triturated withether:hexanes 1:1 to give the desired acid (114 mg, 65%) as a whitesolid. MS (M−OH)⁺=385.

Step (3e): Following a procedure analogous to the preparation ofcompound of Step (1g), but using compound from Step (3d) (82 mg, 0.20mmoles) the title compound was prepared. The crude product was purifiedby reverse phase HPLC on a Vydac C-18 column to give the desired product(80 mg, 94%). MS (M−H)⁻=416.

Example 4 (2R,3S)N1-[(3S)-hexahydro-1-(3-phenoxybenzyl)-2-oxo-1H-azepin-3-yl]-N4-(methyl)-2-(2-methylpropyl)-3-(propyl)-butanediamide

Following a procedure analogous to the preparation of Example 3,compound of Step (2e) (100 mg, 0.20 mmol) was treated with HATU(O-(7-azabenzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumhexafluorophosphate) (114 mg, 0.30 mmol) and N-methyl morpholine (66 mL,0.6 mmol) in 2 mL of DMF for 15 min at rt. A solution of 2.0 Mmethylamine in THF (0.2 mL, 0.4 mmol) was added and the reactionsolution was stirred for 1 h at rt. The reaction solution was dilutedwith 1N HCl (5 mL) and extracted 3× with 10 mL of ethyl acetate. Thecombined organic layers were washed with a saturated sodium bicarbonatesolution (5 mL) and brine (5 mL), dried over magnesium sulfate, andconcentrated in vacou to provide the crude amide. Purification byreverse phase HPLC on a Vydac-18 column provided the desired amide (30mg, 30%). MS (M+Na)⁺=544.

Example 5 (2R,3S)N1-[(3S)-hexahydro-1-(3-phenoxybenzyl)-2-oxo-1H-azepin-3-yl]-N4-(methoxy)-N4-(methyl)-2-(2-methylpropyl)-3-(propyl)-butanediamide

Following a procedure analogous to the preparation of Example 4,compound of Step (2e) (100 mg, 0.20 mmol) was activated and condensedwith N,O-dimethylhydroxylamine hydrochloride (40 mg, 0.40 mmol).Purification by reverse phase HPLC on a Vydac-18 column provided thedesired amide (30 mg, 30%). MS (M+Na)⁺=574.

Example 6 (2R,3S)N1-[(3S)-hexahydro-1-(3-phenoxybenzyl)-2-oxo-1H-azepin-3-yl]-N4-(methoxy)-2-(2-methylpropyl)-3-(propyl)-butanediamide

Following a procedure analogous to the preparation of Example 4,compound of Step (2e) (100 mg, 0.20 mmol) was activated and condensedwith O-methylhydroxylamine hydrochloride (40 mg, 0.40 mmol).Purification by reverse phase HPLC on a Vydac-18 column provided thedesired amide (30 mg, 30%). MS (M+Na)⁺=560.

Example 7 (2R,3S)N1-[(3S)-hexahydro-1-(3-phenoxybenzyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

Following a procedure analogous to the preparation of Example 4,compound of Step (2e) (100 mg, 0.20 mmol) was activated and condensedwith a 2.0 M solution of ammonia in dioxane (0.2 mL, 0.4 mmol).Purification by reverse phase HPLC on a Vydac-18 column provided thedesired amide (30 mg, 30%). MS (M+Na)⁺=530.

Example 7T

Example 7T was synthesized by reducing the double bond present in thecompound of Example 8. Thus, the compound of Example 8 was dissolved intetrahydrofuran and hydrogenated using tritium gas, by methods known toone skilled in the art organic synthesis. Purification by reverse phaseHPLC on a Vydac-18 column provided the desired tritiated amide Example7T wherein m is approximately 2.

Example 8 (2R,3S)N1-[(3S)-hexahydro-1-(3-phenoxybenzyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

Example 8 was synthesized following a procedure analogous to thepreparation of Example 7, but using succinate 9 (Scheme 2). The compoundwas purified by chromatography eluting with 5% methanol in CH₂Cl₂ toafford approx. 500 mg of Example 8. MS (M+Na)⁺=528.

Example 9 (2R,3S)N1-[(3S)-hexahydro-1-(3-phenoxybenzyl)-2-oxo-1H-azepin-3-yl]-N4-(hydroxy)-2-(2-methylpropyl)-3-(allyl)-butanediamide

Example 9 was synthesized following a procedure analogous to thepreparation of Example 2, but using succinate 9 (Scheme 2). Purificationby reverse phase HPLC on a Vydac-18 column provided 150 mg of Example(9). MS (M+Na)⁺=544.

Example 10 (2R,3S)N1-[(3S)-hexahydro-1-(benzophenon-3-yl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(allyl)-butanediamide

(Step 10-a): 3-Bromomethylbenzophenone. A solution of3-methylbenzophenone (20 g, 102 mmol) dissolved in 40 mL of1,2-dibromoethane was heated to reflux. Over a period of about 3 hours asolution of 105 mmol of bromine dissolved in 6 mL of 1,2-dibromoethanewas added to the refluxing solution. After the addition was complete thesolution was allowed to cool to rt and diluted with 100 mL ofdichloromethane. The organic layer was extracted with 1×25 mL of 1 NHCl, 2×15 mL of NaHCO₃ Solution, and 2×25 ML of brine. The organiclayers were dried over magnesium sulfate and concentrated in vacuo. Theresidue was then distilled to afford the product, 16.5 g (60 %) as anoil that solidified upon standing, b.p. 160° C. at 300 mTorr. ¹H NMRanalysis shows that the product contains approximately 7% of thedibromide.

Step (10-b):3-(1,1-dimethylethylcarbomethoxy-N-(benzophenone-3-yl-methyl)caprolactam.Diisopropylamine (4.2 mL, 30 mmol) was dissolved in 25 mL of THF andchilled to −78° C. To the solution was added 10 mL of 2.5Mn-butyllithium in hexanes and the solution was warmed to 0° C. andallowed to stir for 10 min. A solution of Boc-protected aminocaprolactamla (5.0 grams, 22 mmol) dissolved in 25 mL of THF was then added and thereaction solution was stirred for 1 h at 0° C. Solid3-bromomethyl-benzophenone was then added and the reaction solution wasallowed to warm to rt and stir overnight. The reaction solution wasdiluted with water and extracted into ethyl acetate (100 mL). Theorganic layer was rinsed with 2×25 mL of 1 N HCl, 2×25 mL of saturatedNaHCO₃ and 2×25 mL of brine, dried over magnesium sulfate, and dried invacuo. Chromatography eluting with a gradient of 30% to 40% ethylacetate in hexanes afforded the pure benzophenone-substitutedcaprolactam derivative (7.4 g, 80%). MS (M+Na)⁺=445.

The title compound, Example 10, was synthesized in a manner analagous tothe synthesis of the compound of Example 8 using succinate 9 and thebenzophenone-substituted caprolactam derivative of the previous step.The compound was purified by crystallization from ethyl acetate toafford 0.26 g of crystals. MS (M+Na)⁺=540.

Example 11 (2R,3S)N1-[(3S)-hexahydro-1-(benzophenon-3-yl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

The compound of Example 11 was synthesized in a manner analagous to thesynthesis of the compound of Example 8 using succinate 10 and thebenzophenone-substituted caprolactam derivative of Step (10-b). Thecompound was purified by crystallization from ethyl acetate to afford0.25 g of crystals. MS (M+Na)⁺=542.

Example 11T

Example 11T was synthesized by reducing the double bond present in thecompound of Example 10. Thus, the compound of Example 11T was dissolvedin tetrahydrofuran and hydrogenated using tritium gas, by methods knownto one skilled in the art organic synthesis. Purification by reversephase HPLC on a Vydac-18 column provided the desired tritiated amideExample 11T wherein m is approximately 2.

Example 13 (2R,3S)N1-[(3S)-hexahydro-1-(3-(4-fluorophenyl)benzyl)-2-oxo-1H-azepin-3-yl]-N4-(hydroxy)-2-(2-methylpropyl)-3-(propyl)-butanediamide

The general procedure reported for Scheme 5 was followed using4-fluorophenyl boronic acid. Purification afforded 5.0 mg (54%) of thedesired product. MS (M+Na)⁺=548.

Example 16 (2R,3S)N1-[(3S)-hexahydro-1-(3-(3-methylphenyl)benzyl)-2-oxo-1H-azepin-3-yl]-N4-(hydroxy)-2-(2-methylpropyl)-3-(propyl)-butanediamide

The general procedure reported for Scheme 5 was followed using3-methylphenyl boronic acid. Purification afforded 3.0 mg (33%) of thedesired product. MS (M+Na)⁺=544.

Example 22 (2R,3S)N1-[(3S)-hexahydro-1-(3-(2-naphthyl)benzyl)-2-oxo-1H-azepin-3-yl]-N4-(hydroxy)-2-(2-methylpropyl)-3-(propyl)-butanediamide

The general procedure reported for Scheme 5 was followed using2-naphthyl boronic acid. Purification afforded 3.0 mg (31%) of thedesired product. MS (M+Na)⁺=580.

It will be understood by one skilled in the art that Scheme 6 can befollowed in a manner analogous to the procedure for Scheme 5.

Example 23 (2R,3S)N1-[(3S)-hexahydro-1-(3-(4-methoxyphenyl)benzyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

The general procedure reported for Scheme 6 was followed using4-methoxyphenyl boronic acid. Purification afforded 0.5 mg of thedesired product. MS (M+Na)⁺=544.

Example 24 (2R,3S)N1-[(3S)-hexahydro-1-(3-(3-fluorophenyl)benzyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

The general procedure reported for Scheme 6 was followed using3-fluorophenyl boronic acid. Purification afforded 1.6 mg of the desiredproduct. MS (M+Na)⁺=532.

Example 25 (2R,3S)N1-[(3S)-hexahydro-1-(3-(4-trifluoromethylphenyl)-benzyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

The general procedure reported for Scheme 6 was followed using4-trifluoromethylphenyl boronic acid. Purification afforded 2.0 mg (40%)of the desired product. MS (M+Na)⁺=582.

Example 26 (2R,3S)N1-[(3S)-hexahydro-1-(3-(4-methoxyphenyl)benzyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

The general procedure reported for Scheme 6 was followed using4-methoxyphenyl boronic acid. Purification afforded 0.5 mg of thedesired product. MS (M+Na)⁺=544.

Example 27 (2R,3S)N1-[(3S)-hexahydro-1-(3-(2,4-dichlorophenyl)benzyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

The general procedure reported for Scheme 6 was followed using2,6-dichlorophenyl boronic acid. Purification afforded 1.8 mg (11 %) ofthe desired product. MS (M+Na)⁺=582.

Example 28 (2R,3S)N1-[(3S)-hexahydro-1-(3-(4-methylphenyl)benzyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

The general procedure reported for Scheme 6 was followed using 4-tolylboronic acid. Purification afforded 1.8 mg (12 %) of the desiredproduct. MS (M+Na)⁺=528.

Example 29 (2R,3S)N1-[(3S)-hexahydro-1-(3-(3-chloro-4-fluorophenyl)-benzyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

The general procedure reported for Scheme 6 was followed using4-fluoro-3-chlorophenyl boronic acid. Purification afforded 0.5 mg(3.3%) of the desired product. MS (M+Na)⁺=567.

Example 30 (2R,3S)N1-[(3S)-hexahydro-1-(3-(3-methoxyphenyl)benzyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

The general procedure reported for Scheme 6 was followed using2-methoxyphenyl boronic acid. Purification afforded 0.8 mg (5.3%) of thedesired product. MS (M+Na)⁺=544.

Example 31 (2R,3S)N1-[(3S)-hexahydro-1-(3-(2-methoxyphenyl)benzyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

The general procedure reported for Scheme 6 was followed using2-methoxyphenyl boronic acid. Purification afforded 1.5 mg (10%) of thedesired product. MS (M+Na)⁺=544.

It will be understood by one skilled in the art that Scheme 7 can befollowed in a manner analogous to the procedure for Schemes 5 and 6.

Example 32(2R,3S)N1-[(3S)-hexahydro-1-(3-(4-methoxyphenyl)pyrid-5-ylmethyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

Amide 35 of Scheme 7 (0.10 g, 0.18 mmol) was dissolved in 5 mL oftoluene and 41 mg (0.27 mmol) of 4-methoxyphenyl boronic acid was added,followed by 31 mg (0.0147 mmol) oftetrakis(triphenylphosphine)palladium, 0.5 mL of a 2M sodium cabonatesolution and 0.5 mL of methanol. The reaction solution was heated toreflux for 16 h and then allowed to cool to rt. The reaction solutionwas diluted with 10 mL of water and extracted 2× with 50 mL of ethylacetate. The combined organic layers were dried and concentrated and theresulting oil was purified by chromatography eluting with 30 to 100%ethyl acetate in hexanes as a solvent to provide 30 mg (29%) of biarylproduct. MS (M+H)⁺=580.

The purified biaryl product was dissolved in 10 mL of 1:1trifluoroacetic acid/CH₂Cl₂ and stirred at rt for 2 h. The solvents werethen removed under reduced pressure and the resulting oil wasredissolved in 5 mL of toluene and reconcentrated to remove residualTFA. The crude acid (25 mg, 0.047 mmol) was then dissolved in 1 mL ofDMF and 10 μL of N-methylmorpholine (0.094 mmol) and 42 mg (0.062 mmol)HATU were added and the reaction solution was stirred at rt for 45 min.Gaseous ammonia was then bubbled in at a gentle rate for about 1 minuteand the solution was stirred for an additional 1 min. The reactionsolution was then diluted with 10 mL of water and extracted 3× with 30mL of ethyl acetate. The combined organic layers were dried andconcentrated under reduced pressure to a solid which was purified byreversed phase HPLC to provide 3.5 mg (10%) of the compound of Example30 as its trifluoroacetic acid salt. MS (M+H)⁺=523.

Example 33 (2R,3S)N1-[(3S)-hexahydro-1-(3-(4-trifluoromethylphenyl)pyrid-5-ylmethyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

The general procedure reported for the compound of Example 32 wasfollowed using 4-trifluoromethylphenyl boronic acid. Purification byHPLC afforded 6.0 mg of the desired product from as its trifluoroaceticacid salt. MS (M+Na)⁺=583.

Example 34 (2R,3S)N1-[(3S)-hexahydro-1-(3-(3-chloro-4-fluorophenyl)pyrid-5-ylmethyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

Amide 35 (0.30 g, 0.54 mmol) was dissolved in 3 mL of DMF and 123 mg(0.70 mmol) of 4-methoxyphenyl boronic acid was added, followed by 44 mg(0.0543 mmol) of bis(diphenylphosphinoferrocene) palladium (II)dichloride and 1.0 mL (7.18 mmol) of triethylamine. The reactionsolution was heated to 80° C. for 24 h and then allowed to cool to rt.The reaction solution was diluted with 10 mL of water and extracted 2×with 50 mL of ethyl acetate. The combined organic layers were dried andconcentrated and the resulting oil was purified by chromatographyeluting with 20 to 100% ethyl acetate in hexanes as a solvent to provide140 mg (50%) of biaryl product. MS (M+Na)⁺=624.

The general procedure reported for the compound of Example 32 was thenfollowed to provide the amide. Purification by chromatography elutingwith 20 to 100% ethyl acetate in hexanes afforded 45 mg of the desiredproduct of Example 34 as its trifluoroacetic acid salt. MS (M+Na)⁺=567.

Example 39 (2R,3S)N1-[(3S)-hexahydro-1-(4-(4-trifluoromethylphenyl)-benzyl)-2-oxo-1H-azepin-3-yl]-2-(2-methylpropyl)-3-(propyl)-butanediamide

Step (39-a)3-(1,1-dimethylethylcarbomethoxy-N-(4-bromophenylmethyl)caprolactam.

The title compound was synthesized in a manner analogous to thepreparation of3-(1,1-dimethylethylcarbomethoxy-N-(benzophenone-3-yl-methyl)caprolactamin Example 10 but using 4-bromobenzyl bromide as the alkylating agent.The compound was purified by chromatography eluting with 5-20% ethylacetate in hexanes as eluent to provide 7.0 g (70%) of the titlecompound as a solid. MS (M+Na)⁺=419.

Step (39-b)3-(1,1-dimethylethylcarbomethoxy-N-(4,-(4′-trifluoromethylphenyl)phenylmethyl)caprolactam.

To a solution of3-(1,1-dimethylethylcarbomethoxy-N-(4-bromophenylmethyl)caprolactam (0.5g, 1.26 mmol) dissolved in 10 mL of toluene was added 263 mg (1.38 mmol)of 4-trifluoromethylphenyl boronic acid, 1 mL of methanol, and 1 mL of a2M solution of potassium carbonate. The solution was degassed bynitrogen bubbling for 5 min, and then 33 mg oftris(dibenzylideneacetone)dipalladium(0) chloroform adduct and 66 mg oftriphenylphosphine was added. The solution ws heated to reflux for 16 hand then allowed to cool and diluted with 20 mL of water. The aqueouslayer was extracted 3× with 25 mL of ethyl acetate and concentrated. Theresulting oil was purified by chromatography eluting with 20% ethylacetate in hexanes to afford 0.47 g (81%) of an oil which crystallizedon standing.

Step (39-d) The title compound, Example 39, was synthesized in a manneranalagous to the synthesis of the compound of Example 8 using succinate10 (280 mg, 1.04 mmol) and3-(1,1-dimethylethylcarbomethoxy-N-(4,-(4′-trifluoromethylphenyl)-phenylmethyl)caprolactam.The compound was purified by chromatography eluting with 20-100% ethylacetate in hexanes to afford 40 mg of a white powder. MS (M+H)⁺=560.

Example 40 (2S,3R)N1-[(3S)-hexahydro-1-(3-(2-tetrazolylphenyl)benzyl)-2-oxo-1H-azepin-3-yl]-2-(propyl)-3-(2-methylpropyl)-butanediamide

Step (40-a): The compound of Example 40 was synthesized in a manneranalogous to the synthesis of the compound of Example 39, but using thesubstituted acid 28 of Scheme 6 (50 mg, 0.10 mmol) ando-((N-trityl)-tetrazole)phenylboronic acid under the conditions for theformation of the compound (39-b). The desired biaryl acid was isolatedas an impure mixture (134 mg) and used directly in Step (40-b).

Step (40-b): The acid from Step (40-a) (134 mg, impure mixture) wasconverted to the amide under the conditions reported for the compound ofExample 7. The crude amide was then dissolved in 2 mL of 10%trifluoroacetic acid in methanol and allowed to stir at rt for 30 min.The solvents were removed and the residue was purified by chromatographyeluting with 10% methanol in ethyl acetate to provide 40 mg (71%, 2steps) of the compound of Example 40 as a sticky powder. MS (M+Na)+=582.

Example 41 (2S,3R)N1-[(3S)-hexahydro-1-(3-phenoxybenzyl)-2-oxo-1H-azepin-3-yl]-2-(propyl)-3-(2-methylpropyl)-butanediamide

Step (41-a): The compound of Example 41 is formed by coupling Succinate23 (480 mg, 1.21 mmol) with the substituted caprolactam TFA salt 2cunder the conditions reported for the synthesis of the compound ofExample 8. The crude fluorenylmethyl ester was used in the next stepwith out further purification. MS (M+Na)+=709.

Step (41-b): The crude fluorenylmethyl ester is dissolved in 2 mL of a50% solution of piperidine in CH₂Cl₂ and stirred for 3 h at rt. A 10 mLportion of 1N HCl was then added and the mixture was extracted 3× with10 mL of ethyl acetate. The crude acid was used in the next step without further purification. MS (M+H)+=509.

The compound of Example 41 was then prepared using the acid from Step(41-b) under the conditions reported for compound of Example 7. Thecompound was purified by chromatography eluting with 5% methanol inCH₂Cl₂ to afford 120 mg (19%, 3 steps) of a white powder. MS (M+H)+=508.

Example 42(2S,3R)N1-[1,3-dihydro-1-(3-phenoxybenzyl)-2-oxo-5-(phenyl)-2H-1,4-benzodiazepin-3-yl]-2-(2-methylpropyl)-3-(allyl)-butanediamide

Step (42-a) 3-Phenoxybenzyl Iodide:

To a solution of 3-phenoxybenzyl chloride (10.0 g, 45.7 mmol) in 200 mlacetone was added sodium iodide (7.6 g, 507 mmol). The mixture wasstirred at temperature overnight. The mixture was diluted with 300 mlhexane and the organic layer was washed twice with 5% sodiumbicarbonate, once with brine and then dried over MgSO₄. Evaporation ofthe filtrate gave a light yellow oil. The product was used in next stepwithout purification. ¹H NMR (CDCl₃) 4.4 (s,2H), 6.8-7.4 (m, 9H).Step (42-b):

To a solution of benzodiazepine 50 (910 mg, 3.63 mmol), succinate 9 (980mg, 3.63 mmol), hydroxybenzotriazole (980 mg., 7.25 mmol) and EDC (870mg, 4.54 mmol) in 100 ml CH₂Cl₂ at 0 degrees was added triethylamine(0.76 ml, 5.45 mmol). The reaction mixture was washed with saturatedsodium bicarbonate solution, 1.0N HCl, brine and dried over MgSO₄.Evaporation of the organic layer and purification by columnchromatography on silica gel with hexane-ethyl acetate (7:3) gave 610 mgof benzodiazepine 51 as a white solid. M+H=504.37. ¹H NMR (CDCl₃)0.8-1.0 (m, 6H), 1.0-1.2 (m, 1H), 1.4-1.5 (d, 9H), 1.6-1.9 (m, 2H),2.2-2.8 (m, 4H), 4.9-5.2 (m, 2H), 5.6 (dd, 1H), 5.6-6.0 (m, 1H), 7.0-7.6(m, 9H).Step (42-c):

To a solution of benzodiazepine 51 (440 mg, 0.875 mmol) in DMF (20 ml)at 0 degrees was added NaH (45 mg, 1.12 mmol). The mixture was stirredat 0 degrees for 1.5 hr and then a solution of 3-phenoxylbenzyl iodide(330 mg, 1.06 mmol) in 10 ml DMF was added dropwise. The reactionmixture was allowed to warm to room temperature and stirred overnight.TLC using hexanes:EtOAc 6:4 (product Rf=0.31) indicated that thereaction was complete. The reaction mixture was quenched with water, andthe solvent was evaporated under high vacuum, which provided a viscousyellow oil. The product benzodiazepine 52 was dissolved in ethylacetate, which was washed with water (2×), brine and then dried overMgSO₄.

Evaporation of solvent gave 600 mg of benzodiazepine 52 as a yellow oilwhich was not further purified. M+H=686.3, M+Na=708.3. ¹H NMR (CDCl₃)0.8-1.0 (m, 6H), 1.0-1.3 (m, 1H), 1.4-1.5 (d, 9H), 1.5-1.9 (2H), 2.2-2.7(4H), 4.6-4.8 (d,1H), 4.9-5.2 (m, 2H), 5.6-5.9 (m, 3H), 6.6-7.6 (m,18H).

A solution of benzodiazepine 52 in 40 ml of TFA/CH₂Cl₂ (1:1) was stirredovernight at room temperature then evaporated to dryness. Repeatedaddition of toluene and evaporation provided 560 mg. of 53 as a yellowsolid. (M−H=629.1)Step (42-d):

To a solution of benzodiazepine 53 and HATU (410 mg, 1.08 mmol) in 30 mlDMF was added diisopropylethylamine (0.6 ml, 3.44 mmol) at 0 degrees.After 10 minutes, ammonia gas was bubbled through the solution for twominutes, and the reaction mixture was allowed to warm to roomtemperature and stirred overnight. Addition of water and solventevaporation under high vacuum provided a yellow solid. The solid wastaken up in ethyl acetate-water (1:1), and the organic layer was washedwith water (2×), brine and then dried over MgSO₄. Evaporation of solventgave a light yellow solid. Chromatographic purification on silica gelusing CH₂Cl₂ : methanol (10:0.5) gave 256 mg of Example 42. M+H=629.2HNMR (CDCl₃) 0.8-1.0 (m, 6H), 1.2-1.4 (m, 1H), 1.6-2.0 (m, 2H),2.2-2.8(4H), 4.6-4.8 (m, 1H), 5.0-5.2(m, 2H), 5.6-5.9 (m, 3H), 6.2-7.8(m, 18H).

Example 43 (2S,3R)N1-[1,3-dihydro-1-methyl-2-oxo-5-(phenyl)-2H-1,4-benzodiazepin-3-yl]-2-(2-methylpropyl)-3-(allyl)-butanediamide

Step (43-a):

A solution of tert-butyl succinate ester 9 (1.1 eq.) in DMF (0.25 M)under N₂ at 0° C. was added HATU ( 1.1 eq.), then Hunig's base (4.0eq.). The mixture was stirred at 0° C. for 10 mins. A solution of2,3-dihydro-1-methyl-3-amino-5-phenyl-1H-1,4-benzodiazepin-2-one 54 inDMF (0.8 M) (1.0 eq.) was added to this solution. The reaction mixturewas stirred overnight at room temperature and then transfered to aseparatory funnel containing water. 30% n-Hexane in ethyl acetate wasadded which gave a clear organic layer. The aqueous solution wasextracted twice with 30% n-hexane in ethyl acetate. The combined organiclayers were washed with water and brine, dried over magnesium sulfate,and concentrated in vacuo. The residue was purified by chromatography onflash grade silica gel using 20% ethyl acetate in n-hexane. The compound55 was isolated as an amorphous white solid (85%). Rf=0.25 (7:3n-hexane:ethyl acetate).

¹H-NMR:(CDCl₃): δ 7.61-7.21 (m, 10H); 5.77-5.73 (m, 1H); 5.57-5.54 (d,1H); 5.20-4.97 (m, 2H); 3.47 (s, 3H); 2.63-2.33 (m, 4H); 1.80-1.76 (m,2H); 1.47-1.46 ( d, 9H); 1.43-1.11 (m, 1H); 1.01-0.86 (m, 6H). MS:C₃₁H₃₉N₃O₄ (M+H) 518.3 (M+Na) 540.3.Step (43-b):

A solution of 55 in 50% TFA in methylene chloride (0.15M) was stirred atroom temperature overnight. The solution was concentrated in vacuo,washed and concentrated four times with toluene in vacuo to givecompound 56 as an amorphous solid (95%). Rf=0.64 (9.5 : 0.5 methylenechloride : methanol). MS: C₂₇H₃₁N₃O₄ (M+H) 462.Step (43-c):

To a solution of 56 (1.0 eq.) in DMF (0.25 M) under N₂ at 0° C. wasadded HATU (1.1 eq.), and then Hunig's base (4.0 eq.). The mixture wasstirred at 0° C. for 10 mins, and then anhydrous ammonia bubbled throughthe solution for two minutes. The reaction mixture was stirred overnightat room temperature and then transfered to a separatory funnelcontaining water and diluted with 30% n-hexane in ethyl. The aqueoussolution was extracted twice with 30% n-hexane in ethyl acetate. Thecombined organic layers were washed with water and brine, dried overmagnesium sulfate, and concentrated in vacuo. The residue was purifiedby chromatography on flash grade silica gel using 4% methanol inmethylene chloride. The title compound, Example 43, was isolated as anamorphous white solid (87%). Rf=0.43 (9:1 methylene chloride:methanol).

¹H NMR:(CDCl₃): δ 7.63-7.22 (m, 10H); 6.25-6.13 (d, 1H); 5.88-5.73 (m,1H); 5.53-5.51 (dd, 1H); 5.44-5.41 (d, 1H); 5.22-5.04 (m, 2H); 3.47-3.46(d, 3H); 2.74-2.31 ( m, 4H); 1.81-1.61 (m, 2H); 1.34-1.22 (m, 1H);0.99-0.87 (m, 6H). MS: C₂₇H₃₂N₄O₃ (M+H) 461.

It is understood that the (R) or (S)-benzodiazepine diastereomer ofExample 43 can be prepared using methods analogous to the presentexample but employing the (R) or (S) stereoisomer of intermediate 2a inStep (43-a), respectively.

Example 43T Tritiated (2S,3R)N1-[1,3-dihydro-1-methyl-2-oxo-5-(phenyl)-2H-1,4-benzodiazepin-3-yl]-2-(2-methylpropyl)-3-(n-propyl)-butanediamide

Example 43T was synthesized by reducing the double bond present in the(S)-benzodiazepine diastereomer of Example 43. The (S) diastereomer ofExample 43 may be separated from the product of Step (43-c) by meansknown to one skilled in the art and the single isomer reduced.Alternatively, this diastereomer may be prepared directly as statedabove. Thus, the (S)-benzodiazepine diastereomer of Example 43 wasdissolved in tetrahydrofuran and hydrogenated using tritium gas, bymethods known to one skilled in the art organic synthesis. Purificationby reverse phase HPLC on a Vydac-18 column provided the desiredtritiated amide Example 43T wherein m is approximately 2.

It is understood that one skilled in the art of organic synthesis cansynthesize radiolabeled compounds of the present invention for use as atagged inhibitor of beta-amyloid production using radiolabelingtechniques well know in the art. For example tritiation, using catalystssuch as Pd/C or Wilkinson's catalyst and ³H₂ gas, one skilled in the artcan reduce olefin precursors. Examples of olefin precursors are Examples8, 10, 42, 43, intermediate Succinate 10 and intermediate Benzodiazepine51.

Representative Procedures for the Synthesis of Radiolabeled Compounds

Described below are representative procedures for the synthesis ofradiolabeled compounds. These nonlimiting representative procedures, andother procedures known in the art, will be readily known and appreciatedby one of skill in the art of organic synthesis of radiolabeledcompounds. The radiolabeled ligands of macromolecules involved in theprocess of APP and/or beta-amyloid production (the “radiolabeledligands”) of the present invention can be synthesized using standardsynthetic methods known to those skilled in the art, using radioisotopesof halogens (such as chlorine, fluorine, bromine and iodine), as well asothers. Radioisotopes include ¹²³I, ¹²⁵I, ¹³¹I, ^(99m)Tc, and ¹¹¹In.

The radiolabeled ligands of the invention may be labeled either directly(that is, by incorporating the radiolabel directly into the compounds)or indirectly (that is, by incorporating the radiolabel into thecompounds through a chelator which has been incorporated into thecompounds. For brain imaging, it is expected that direct labeling willbe preferred in the present invention. For direct labeling, as thoseskilled in the art will recognize, the labeling may be isotopic ornonisotopic. With isotopic labeling, one group already present in thecompound is substituted with (exchanged for) the radioisotope. Withnonisotopic labeling, the radioisotope is added to the compound withoutsubstituting with (exchanging for) an already existing group.

Generally, labeled compounds are prepared by procedures which introducethe labeled atom at a late stage of the synthesis. This allows formaximum radiochemical yields, and reduces the handling time ofradioactive materials. When dealing with short half-life isotopes, amajor consideration is the time required to conduct syntheticprocedures, and purification methods. Protocols for the synthesis ofradiopharmaceuticals are described in Tubis and Wolf, Eds.,“Radiopharmacy”, Wiley-Interscience, New York (1976); Wolf, Christman,Fowler, Lambrecht, “Synthesis of Radiopharmaceuticals and LabeledCompounds Using Short-Lived Isotopes”, in Radiopharmaceuticals andLabeled Compounds, Vol 1, p. 345-381 (1973), the disclosures of each ofwhich are hereby incorporated herein by reference, in their entirety.

Various procedures may be employed in preparing the radiolabeledcompounds of the invention where the radiolabel is a halogen. Somecommon synthetic methodologies for isotopic halogen labeling of aromaticcompounds such as the type present here are iododediazonization,iododeborobation, iododestannylation, iododesilation, iododethallation,and halogen exchange reactions. The most common synthetic methodologyfor nonisotopic halogen labeling of aromatic compounds such as the typepresent here is iododeprotonation or electrophilic aromatic substitutionreactions. These methods and additional procedures are described inMerkushev, Synthesis, 923 (1988), and Seevers et al., Chem. Rev., 82:575 (1982), the disclosures of each of which are hereby incorporatedherein by reference, in their entirety.

By way of example, isotopically radiolabeled 4, 5 and 6-halot-butyloxycarbonyl-3-aminomethylbenzoic acid derivatives may be preparedusing the general procedures described above for the synthesis of theunlabeled compounds. In carrying out such radiolabeling, it is importantthat the half-life of the isotope chosen be much longer than thehandling time of the reaction sequences. Known starting materialsinclude the 2, 3, and 4-iodo (123I, 125I, and 131I) benzoic acids.Iodo-radiolabeled compounds may also be isotopically prepared fromanilines by the Sandmeyer reaction as described in Ellis et al., Aust.J. Chem., 26: 907 (1973).

Alternatively, radiolabeled compounds may prepared by way of isotopiclabeling from an unlabeled bromo or iodo derivatives by various two stepreaction sequences, such as through the use of trialkylsilyl synthons asdescribed in Wilson et al., J. Org. Chem., 51: 483 (1986) and Wilbur etal., J. Label. Compound. Radiopharm., 19: 1171 (1982), the use oftrialkylsilyl synthons as described in Chumpradit et al. J. Med. Chem.,34: 877 (1991) and Chumpradit et al J. Med. Chem., 32: 1431 (1989), andthe use of boronic acid synthons as described in Kabalka et al., J.Label. Compound. Radiopharm., 19: 795 (1982) and Koch et al., Chem.Ber., 124:2091 (1991).

In preparing radiolabeled compounds of the present invention, tomaximize radiochemical yields, to reduce the handling time ofradioactive materials, and to prepare short half-life halogen labeledcompounds, it is preferable to perform the isotopic halogen labeling asone of the final steps in the compound synthesis. The following providesexemplary proceudres for such late stage labeling.

Unlabeled iodo compounds are versatile precursors which can be convertedto the labeled derivatives by any of the two step reaction sequencesdescribed above. In general, useful functionalities to incorporate intoa compound includes bromo, the nitro, the trialkylsilyl, thetrialkyltin, and the boronic acid groups. The synthesis and applicationof each of these precursors is described above. Radioiodination of acompound of the present invention may be achieved via isotopic labelingduring the final stages of preparation by the substitution ofradioactive iodide for a stable iodine atom already present in themolecule. This can often be done by heating the compound withradioactive iodide in an appropriate solvent as described in Ellis etal., Aust. J. Chem., 26: 907 (1973).

In some cases radiolabeled compounds of the present invention may alsobe isotopically iodo-labeled during the final stages of theirpreparation from anilines by the Sandmeyer reaction as described inEllis et al., Aust. J. Chem., 26: 907 (1973). Alternatively, a compoundmay be isotopically labeled late in the reaction scheme from theunlabeled bromo or iodo derivatives by various two step reactionsequences, as described above, such as through the use of trialkylsilylsynthons as described in Wilson et al., J. Org. Chem., 51: 4833 (1986)and Wilbur et al., J. Label. Compound. Radiopharm., 19: 1171 (1982),through the use of trialkylsilyl synthons as described in Chumpradit etal., J. Med. Chem., 34: 877 (1991) and Chumpradit et al., J. Med. Chem.,32: 1431 (1989), and through the use of boronic acid synthons asdescribed in Kabalka et al., J. Label. Compound. Radiopharm., 19: 795(1982) and Koch et al., Chem. Ber., 124:2091 (1991).

A related approach where the isotopic halogen radiolabeling may becarried out late in the synthesis scheme involves converting a syntheticintermediate compound that already incorporates a trialkylsilyl,trialkyltin, or boronic acid groups. Labeled iodo derivatives may alsobe readily prepared nonisotopically from the amino, hydroxy, or methoxysubstituted compounds as described in Arora et al J. Med. Chem., 30:918(1987). Electrophilic aromatic substitution reactions are enhanced bythe presence of such electron-donating substituents.

Another representative approach to the incorporation of a radiolabeledhalogen in compounds containing methyl substituted phenyl involves theconversion to a a-halotoluene derivative with NBS or NCS underfree-radical halogenation conditions. The benzylic halides may besmoothly replaced by radiolabeled iodide through a nucleophilicsubstitution reaction. The above described process chemistry can also beused to prepare any radioactive halogen isotope.

By way of illustration, ¹⁸F derivatives of certain compounds can beprepared by conjugation of ¹⁸F functionalized phenyl intermediates (R.H. Mach et al., J. Med. Chem., 1993, 36,3707-3720).

Utility

Aβ production has been implicated in the pathology of Alzheimer'sDisease (AD). The compounds of the present invention as well ascompounds determined from the present invention have utility for theprevention and treatment of AD by inhibiting the proteolytic activityleading to Aβ production. Methods of treatment target formation of Aβproduction through the enzymes involved in the proteolytic processing ofβ amyloid precursor protein. Compounds that inhibit β or ysecretaseactivity, either directly or indirectly, control the production of Aβ.Such inhibition of β or γ secretases reduces production of Aβ, and isexpected to reduce or prevent the neurological disorders associated withAβ peptide, such as Alzheimer's Disease.

Cellular screening methods for inhibitors of Aβ production, testingmethods for the in vivo suppression of Aβ production, and assays for thedetection of secretase activity are known in the art and have beendisclosed in numerous publications, including PCT publication number WO98/22493, EPO publication number 0652009, U.S. Pat. No. 5,703,129 andU.S. Pat. No. 5,593,846; all hereby incorporated by reference.

The compounds of the present invention as well as compounds determinedfrom the present invention have utility for the prevention and treatmentof disorders involving Aβ production, such as cerebrovascular disorders.

Compounds of Formula (I) are expected to possess γ-secretase inhibitoryactivity. The γ-secretase inhibitory activity of the compounds of thepresent invention is demonstrated using assays for such activity, forexample, using the assay described below. Compounds within the scope ofthe present invention have been shown to inhibit the activity ofγ-secretase, as determined using assays for such activity.

Compounds provided by this invention should also be useful as standardsand reagents in determining the ability of a potential pharmaceutical toinhibit Aβ production. These would be provided in commercial kitscomprising a compound of this invention.

As used herein “μg” or “ug” denotes microgram, “mg” denotes milligram,“g” denotes gram, “μL” denotes microliter, “mL” denotes milliliter, “L”denotes liter, “nM” denotes nanomolar, “μM” or “uM” denotes micromolar,“mM” denotes millimolar, “M” denotes molar, “nm” denotes nanometer,“SDS” denotes sodium dodecyl sulfate, and “DMSO” denotes dimethylsulfoxide, and “EDTA” denotes ethylenediaminetetraacetate.

A compound is considered to be active if it has an IC₅₀ or K_(i) valueof less than about 100 μM for the inhibition of Aβ production orinhibition of proteolytic activity leading to Aβ production. Compounds,as demonstrated by use of the invention, have demonstrated IC₅₀ values,for the inhibition of Aβ production, of less than about 100 μM.Preferably compounds, as demonstrated by use of the invention,demonstrate IC₅₀ values, for the inhibition of Aβ production, of lessthan about 1 μM. More preferably compounds, as demonstrated by use ofthe invention, demonstrate IC₅₀ values, for the inhibition of Aβproduction, of less than about 100 nM. Even more preferably compounds,as demonstrated by use of the invention, demonstrate IC₅₀ values, forthe inhibition of Aβ production, of less than about 50 nM.

β Amyloid Precursor Protein Accumulation Assay (βAPPA Assay)

An assay to evaluate the accumulation of Aβ protein was developed todetect potential inhibitors of secretases. The assay uses the CHO N 9cell line, characterized for expression of exogenous APP byimmunoblotting and immunoprecipitation.

The effect of test compounds on the accumulation of Aβ in theconditioned medium is tested by immunoprecipitation. N 9 cells are grownto confluency in 6-well plates and washed twice with 1× Hank's bufferedsalt solution. The cells are starved in methionine/cysteine deficientmedia for 30 min., followed by replacement with fresh deficient mediacontaining 150 uCi Tran35S-LABEL™ (ICN). Test compounds dissolved inDMSO (final concentration 1%) are added, over a range of 1 picomolar to100 micromolar, together with the addition of the fresh media containingTran35S-LABEL™. The cells are incubated for 4 h at 37° C. in a tissueculture incubator.

At the end of the incubation period, the conditioned medium is harvestedand pre-cleared by the addition of 5 μl normal mouse serum and 50 ul ofprotein A Sepharose (Pharmacia), mixed by end-over-end rotation for 30minutes at 4° C., followed by a brief centrifugation in a microfuge. Thesupernatant is then harvested and transferred to fresh tubes containing5 ug of a monoclonal antibody (examples of antibodies include but arenot limited by, clone 1101.1, directed against an internal peptidesequence in Aβ; or 6E10 from Senetek; or 4G8 from Senetek; additionallypolyclonals from rabbit antihuman Aβ from Boehringer Mannheim) and 50 μlprotein A Sepharose. After incubation overnight at 4° C., the samplesare washed three times with high salt washing buffer (50 mM Tris, pH7.5, 500 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40), three times with lowsalt wash buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5%Nonidet P-40), and three times with 10 mM Tris, pH 7.5. The pellet afterthe last wash is resuspended in SDS sample buffer (Laemmli U.K. Cleavageof structural proteins during the assembly of the head of bacteriphageT4. Nature 227, 680-5, 1970.) and boiled for 3 minutes. The supernatantis then fractionated on either 10-20% Tris/Tricine SDS gels or on 16.5%Tris/Tricine SDS gels. The gels are dried and exposed to X-ray film oranalyzed by phosphorimaging. The resulting image is analyzed for thepresence of Aβ polypeptides. The steady-state level of Aβ in thepresence of a test compound is compared to wells treated with DMSO (1%)alone. A typical test compound in this assay blocks Aβ accumulation inthe conditioned medium, and is considered active with an IC₅₀ less than100 μM.

C-Terminus β Amyloid Precursor Protein Accumulation Assay (CTF Assay)

The effect of test compounds on the accumulation of C-terminal fragmentsis determined by immunoprecipitation of APP and fragments thereof fromcell lysates. N 9 cells are metabolically labeled, as above, with mediacontaining Tran35S-LABEL™, in the presence or absence of test compounds.At the end of the incubation period, the conditioned medium areharvested and cells lysed in RIPA buffer (10 mM Tris, pH 8.0 containing1% Triton X-100, 1% deoxycholate, 0.1% SDS, 150 mM NaCl, 0.125% NaN₃).Again, lysates are precleared with 5 ul normal rabbit serum/50 ulprotein A Sepharose, followed by the addition of BC-1 antiserum (15 μl;)and 50 μl protein A Sepharose for 16 hours at 4° C. Theimmunoprecipitates are washed as above, bound proteins eluted by boilingin SDS sample buffer and fractionated by Tris/Tricine SDS-PAGE. Afterexposure to X-ray film or phosphorimager, the resulting images areanalyzed for the presence of C-terminal APP fragments. The steady-statelevel of C-terminal APP fragments is compared to wells treated with DMSO(1%) alone. A typical test compound in this assay stimulates C-terminalfragment accumulation in the cell lysates, and is considered active withan IC₅₀ less than 100 uM.

Accumulation-Release Assay

This immunoprecipitation assay is specific for γ secretase activity(i.e., proteolytic activity required to generate the C-terminal end ofAβ either by direct cleavage or generating a C-terminal extended specieswhich is subsequently further proteolyzed). N 9 cells are pulse labeledwith media containing Tran35S-LABEL™ in the presence of a reported ysecretase inhibitor (MDL 28170; Higaki J, Quon D, Zhong Z, Cordell B.Inhibition of beta-amyloid formation identifies proteolytic precursorsand subcellular site of catabolism. Neuron 14, 651-659, 1995) for 1 h,followed by washing to remove ³⁵S radiolabel and MDL 28170. The media isreplaced and test compounds are added over a dose range (for example 0.1nM to 100 uM). The cells are chased for increasing periods of times andAβ is isolated from the conditioned medium and C-terminal fragments fromcell lysates (see accumulation assay above). The activity of testcompounds are characterized by whether a stabilization of C-terminalfragments is observed and whether Aβ is generated from these accumulatedprecursor. A typical test compound in this assay prevents the generationof Aβ out of accumulated C-terminal fragments and is considered activewith an IC₅₀ less than 100 μM.

Radioligand Competition Binding Assay (RCB Assay):

The following assay, of the invention, discloses a novel assay torapidly screen and evaluate potential inhibitors of secretases. Theassay enables screening for inhibitors of Aβ production or inhibitors ofproteolytic activity leading to the production of Aβ by using acompetitive binding assay wherein more than one chemical entity competesfor a binding site identified for Aβ production. For example, in acompetitive binding assay of the invention competition occurs betweenpotential Aβ production inhibitors (i.e. compounds being investigatedfor inhibitory activity) and a standard known for Aβ productioninhibitory activity which standard has been tagged by a radiolabel.Example 7T radiolabeled with tritium is a standard identified for Aβproduction inhibitory activity; however, any radiolabelled or taggedcompound binding to the same site as Example 7T could be used in thisassay. It is understood that the theory of competitive binding is wellknown to one skilled in the art of pharmacology. The compoundsidentified by this invention may have utility for the prevention andtreatment of neurological disorders relating to Aβ production, includingAlzheimer's disease, by inhibiting Aβ production.

Materials:

Assay buffer: Hepes 50 mM pH 7.0.

Competing compounds/potential inhibitors: weigh and dilute in 100% DMSOat a concentration of 1×10⁻²M. From that stock, a second (6×10⁻⁴M) stockis made in 100% DMSO. The working stock (6×10⁻⁵M) is made from thesecond in assay buffer containing 6% DMSO.

Wash buffer: Phosphate buffered saline containing 0.01% triton X-100, pH7.0 at 4° C.

Membrane: HEK₂₉₃ control membranes (Receptor Biology, Inc.), or ratwhole brain homogenates prepared as follows: Frozen pellets ofapproximately 10 mg protein HEK₂₉₃ cell membranes are thawed on ice andhomogenized in 10 ml of assay buffer, using a Brinkman Polytron (PT-10)setting 6 for 10 sec. The homogenate was centrifuged at 48,000×g for 12minutes and the resulting pellet washed by repeating the homogenizationand centrifugation steps. The final cell pellet was resuspended inbuffer to yield a protein concentration of approximately 0.35 mg/ml asassayed by the method of Bradford (1976) using bovine serum albumin asthe standard.

Rats: for the rat whole brain homogenates, (male Sprague-Dawley rats 200to 300 g., Charles River) are decapitated and brains dissected on anice-chilled glass plate. Brains weighing ˜2 g are homogenized in 20 mlof assay buffer and prepared by the method described above for the cellhomogenates. The final pellet is resuspended to yield a protein conc. of˜5 mg/ml original wet weight.

Radiolabeled standard: [³H] I-7T (Example 7T; synthesized by DupontPharm. Co.) S.A. 87.5 Ci/mMol, (11.43 μM I-7T).

Radioligand Competition Binding Assay Method:

Assays are initiated by addition of 150 μl membrane suspension (˜0.35 mgprotein/ml) to 150 μl of assay buffer containing 1% DMSO, 5 to 30 nM[³H] I-7T, and various concentrations of inhibitors over a range of 1picomolar to 100 micromolar. Binding assays are preformed in duplicatein disposable polypropylene 96 well plates, (Costar Corp., Cambridge,Mass.) in a final volume of 0.3 ml. Nonspecific binding is defined inthe presence of 3 μM I-7T. Optimum incubation time at 23° C. is 1 hour.The separation of bound radioligand I-7T from free radioligand I-7T isaccomplished by rapid vacuum filtration of the incubation mixture overGFF glass fiber filters (Inotech Biosystems International, Lansing,Mich.) presoaked for 2 hours in 0.3% polyethylinamine (pH 13) using anInotech cell harvester. Filters were washed 2 times with 0.3 ml ofice-cold phosphate buffered saline pH 7.0 containing 0.01% Triton X100.Filters are accessed for radioactivity by liquid scintillation countingusing a Packard 2500 TR (Packard Instrument Co., Downers Grove, Ill.),having a counting efficiency for tritium of ˜56%.

Alternatively, it is well known in the art that a homogenous assayformat, such as a scintillation proximity assay (SPA), could be employedin the radioligand competition binding assay of the invention. Forexample, membranes or membrane extracts can be immobilized onto the SPAsupport, after which the support is then incubated with a taggedinhibitor of beta amyloid production in the presence of a potentialinhibitor of beta amyloid production. The SPA support, by nature of itsconstruction, magnifies the radioactive scintillation signal of boundradioactive compounds while not magnifying the radioactive signal ofradioactive compounds free in solution. Therefore, the bound taggedinhibitor of beta amyloid production is detected and quantified byscintillation counting in the presence of free tagged inhibitor of betaamyloid production.

It is understood that the process of separating bound tagged inhibitorof beta amyloid production from free tagged inhibitor of beta amyloidproduction, for example bound radioligand I-7T from free radioligandI-7T, can be conducted in a number of methods. For example the processof separating includes, but is not limited to, filtration orcentrifugation. The process of separating is intended to facilitatequantification of bound tagged inhibitor of beta amyloid production.Therefore, the process of separating is also intended to encompasshomogeneous techniques, for example SPA, where free tagged inhibitor ofbeta amyloid production in situ is separated from the tagged inhibitorof beta amyloid production bound to the solid support of thescintillant. Thus, in a homogeneous technique such as SPA, the free andbound inhibitors are considered separated from each other within themeaning of the invention.

Radioligand Competition Binding Data Analysis:

Resulting disintigrations per minute (dpm's) are expressed as percentinhibition of [³H] I-7T specific binding. IC₅₀ values of competingcompounds are calculated using the program GraphPad Prism by GraphPadSoftware, (San Diego, Calif.). It is understood that one skilled in theart can determine these values using this program.

A good correlation for inhibition of proteolytic activity leading to Aβproduction has been found between compounds identified in functionalassays for determination of Aβ production, for example the β AmyloidPrecursor Protein Accumulation Assay, and compounds identified in theRadioligand Competitive Binding Assay. The correlation is demonstratedby plotting the IC₅₀ values of compounds identified in the functionalassay verses the IC₅₀ values of compounds identified in the RCB Assay.Compounds from several chemical series, including Examples disclosedherein, have exhibited, over a range of potencies, similar IC₅₀ valuesin the RCB Assay as seen in an accumulation assay.

Example 98

The compound of Example 98 was synthesized according to proceduresdisclosed in PCT Application WO98/28268, published Jul. 2, 1998.

Example 98b

The compound of Example 98b was synthesized according to proceduresdisclosed in PCT Application WO98/28268, published Jul. 2, 1998.

Example 99

Step (99a): The compound of Step (99a) is formed by coupling succinate 7(115 mg, 0.5 mmol) with the substituted caprolactam TFA salt (212 mg,0.5 mmol) from Step (2c) of Example 2 under the conditions reported forthe synthesis of the compound of Example 8. The crude tert-butyl esterwas taken on without further purification.

Step (99b): The compound of Step (99b) is formed by dissolving the crudeproduct from Step (99a) in 5 mL of a 1:1 solution of TFA/CH₂Cl₂ andstirring at room temperature for 2 hours. Concentration followed byreconcentration twice from 10 mL of toluene provides the crude acidwhich was taken on with no further purification.

Step (99c): The title compound, Example 99, was prepared using the acidfrom Step (99b) under the conditions reported for the compound ofExample 7. The compound was purified by chromatography eluting with 5%methanol in CH₂Cl₂ to afford 50 mg (21%, 3 steps) of a white powder. MS(M+Na)⁺=488.

Example 100

Binding of Example 7T to Cell Membranes:

A survey of different cell lines was performed using the radioligandcompetition binding assay, of the invention, with Example 7T to identifymembranes rich in binding sites for Example 7. Cell lines useful forperformance of the RCB Assay are preferentially human or mammalian celllines. It is more prefered that the cell lines express presenilin 1,presenilin 2 and/or presenilin homologs (for example SEL-12). The celllines surveyed included HEK293 cells (ATCC CRL-1573), IMR 32 (ATCCCCL-127), RAJI (ATCC CCL-86), CHO (ATCC CRL-9096), U-937 (ATCCCRL-1593), and THP-1 (ATCC TIB-202). Of the cell lines surveyed the bestsignal to noise ratio (i.e., ratio of specific binding and non-specificbinding) was obtained using THP-1 cell membranes.

Example 101

Characterization of the Example 11T in the Radioligand CompetitionBinding Assay:

Example 11, a benzophenone derivative of Example 7, was synthesized.When Example 11 was assayed in the βAPPA Assay and separately in the RCBAssay with Example 7T as the radiolabeled standard, a statisticallysignificant correlation of IC₅₀ values was observed between the twoAssays.

Radiolabeled Example 11, i.e. Example 11T, was synthesized and tested inthe RCB Assay for equivalency to Example 7T. The apparent Ki wascalculated for four compounds (Example 7, Example 11, Example 98, andExample 99) and an statistically significant correlation was observedbetween results obtained whether the RCB Assay was conducted withExample 7T or Example 11T, indicating that Example 11 binds to the samemolecular target(s) in cell membranes. Therefore, it was found thatExample 11T could be used instead of Example 7T as the radioactivetracer in the RCB Assay.

Analogously, it has also been found that Example 43T can be used insteadof Example 7T as the radioactive tracer in the RCB Assay.

Example 102

Example 11 Reduces the Bmax of Example 7T:

Cell membranes (THP-1) were incubated with Example 11 at approximately 3times the K_(d) concentration for 1 hour at room temperature under theconditions outlined for the RCB Assay. Membranes were photolysed at 365nm for 1 hour on ice. Control membranes were incubated in parallel onice. The membranes were harvested (centrifuged at 40,000 G, 4° C., 20minutes) and extensively washed with assay buffer. The membranes weresubsequently analyzed in the RCB Assay using Example 7T. A bmax of 938fmol/mg membranes was observed for unphotolysed membranes, whereas thebmax was reduced to 238 fmol/mg membranes after photolysing. However,the K_(d) for Example 7T was not statistically significantly changed.These results indicate that Example 11 is cross-linked to the membranebinding site of Example 7.

Bmax is understood by one skilled in the art to represent the maximumnumber of binding sites in a cell membrane. See Mary Keen (Ed.) Receptorbinding techniques. Methods in Molecular Biology, Vol 106, Humana Press,Totowa, N.J., 1999.

In this experiment the membranes were photolysed at 365 nm, which isappropriate for activation of the benzophenone moiety of Example 11. Itis understood that photolysation of the membranes can occur at anywavelength that activates a photoactive tag to cross link to theprotein. Such wavelengths generally occur in the 250 to 450 nm range.

Example 103

Analysis of Cross-Linked Polypeptides by SDS-PAGE:

THP-1 cell membranes were incubated with Example 11T exactly as outlinedunder the methods for the RCB Assay of the invention in the presence ofan unlabeled competing compound; for Example 98 or Example 99. After 1hour incubation at room temperature, the membranes were analyzed by theRCB assay (top panel, FIG. 1). The membranes in parallel wells werephotolysed (365 nm, as in Example 102) for 30 minutes on ice(alternatively, at room temperature). Membranes were collected, boiledin SDS-containing buffer in the absence (middle panel FIG. 1) orpresence of dithiothreitol (50 M) (bottom panel FIG. 1) and fractionatedby SDS-PAGE (12% acrylamide in the separating gel). The polyacrylamidewas fixed in 10% acetic acid/20% methanol/70% water for 45 minutes atroom temperature and soaked for another 45 minutes in AmplifyTM(Amersham). After drying, the gel was exposed to X-ray film. In theabsence of a competing compound, labeling of a number of polypeptideswas observed. However, based on the ability of unlabeled compounds tocompete with the cross-linking reaction, major polypeptides, that couldbe specifically cross-linked with Example 11T, of molecular sizes of 30(band A), 25 (band B), 20 (band C), and 10-12 (band D) kD wereidentified.

Dose-response experiments using increasing concentrations in the rangeof 1 picomolar to 100 micromolar of an unlabeled competing compound wereperformed. The resulting samples were either analyzed by the RCB Assayof the invention prior to photolysis or by SDS-PAGE after photolysis. Astatistically significant correlation was observed between thecompetition in the radioligand competition binding assay and theradioactivity incorporated into the 30 (band A), 25 ( band B), 20kD(band C), and 10-12 kD (band D) bands as revealed by SDS-PAGE andfluorography.

Thus, the quantitative reduction of cross-linking by unlabeled compoundsto the 30, 25, 20, and 10-12 kD bands accurately tracks the reduction ofspecific binding in the binding assay. These results indicate thatidentification of the cross-linked species will identify the site ofinteraction in the binding assay.

FIG. 1 illustrates the correlation between results of the RCB assay andthe cross-linking assay for DMSO (lane 1), Example 7 (lane 2), Example98b (lane 3), Example 43 (lane 4), Example 99 (lane 5), and Example 11(lane 6) at 1 micromolar. FIG. 1, top panel, illustrates results of theRCB Assay for thesae compounds. FIG. 1, middle panel, illustratesresults of the cross-linking assay for these compounds undernon-reducing conditions. FIG. 1, bottom panel, illustrates results ofthe cross-linking assay for these compounds under reducing conditions.Membranes were incubated with Example 11T and DMSO or a number ofunlabeled compounds and analyzed by RCB assay. The total radioactivityassociated with the filter off is indicated. Parallel wells werephotolysed as in Example 103, and the membrane extracts were analyzed bySDS-PAGE followed by fluorography. The mobility of molecular weightmarkers (in kD) is indicated to the right. Note specific cross-linkingto polypeptides of 30 (band A), 25 (band B), 20 (band C), and 10-12(band D) polypeptides. The radioactivity associated with band A isstronger than that in bands B to D, suggesting that band A might be amixture of two polypeptides (i.e., presenilin 1 and presenilin 2; seeFIG. 4).

Example 104

Immunological Identification of the 30 kD and 20 kD Cross-LinkedPolypeptides of Example 103:

THP-1 cell membranes (1 mg/ml) in 50 mM TRIS buffer, pH 7.4-7.5, wereincubated with Example 11T for 1 hour at room temperature and photolysed(as stated above) at room temperature for 30 minutes. The membranes werecollected by centrifugation. The membranes were extracted with 50 mMTris, pH 7.5 containing 100 mM KCl, 2 mM EDTA, 2% CHAPS and 1 completeprotease inhibitor tablet per 25 ml buffer (COMPLETE™, BoehringerMannheim; product number 1697 498)) for 1 hour at 4° C. The detergentsoluble fraction was recovered by centrifugation (40,000 g, 30 min, 4°C.). The membrane extract was diluted one half with water. 500 ul of themembrane extract were pre-incubated with 10 ul of normal mouse IgG and50 ul anti-mouse IgG Sepharose (Sigma) for 1 hour at 4° C. Thesupernatant was recovered by centrifugation. Subsequently, 10 ug ofpreimmune IgG (Sigma) or 10 ug of a monoclonal antibody to presenilin 1was added in the presence of 50 ul anti-mouse IgG Sepharose. Examples ofcommercially available antibodies to presenilin 1 are ChemiconInternational: rat anti-human Presenilin 1 monoclonal antibody; productnumber MAB 1563; or Santa Cruz Biotechnology: goat anti Presenilin 1;product number SC-1244; or Santa Cruz Biotechnology: goat antiPresenilin 1; product number SC-1245. For use with goat antibodies, theimmunoprecipitation was altered as follows: normal goat IgG and proteinG Sepharose was used for the pre-absorption and protein G Sepharose wasused in the presence of the goat antibodies to presenilin 1. Themembrane extract was incubated for 5 hours at 4° C. The Sepharose beadswere collected by centrifugation and washed 3 times with 25 mM Tris, pH7.5 containing 50 mM KCl, 1 mM EDTA and 1% CHAPS, followed by 3 washeswith phosphate buffered saline. Radioactivity bound to the Sepharosebeads was dissociated by boiling in SDS sample buffer (4×) containing 50mM dithiothreitol. The supernatant was loaded onto a 12% SDS-PAGE andthe gel was processed as above. Fluorography revealed the presence ofthe approximately 30 (band A), 20 (band C), and 10 kD (band D)polypeptides in the immunoprecipitation with antibodies to presenilin 1,but not with normal mouse IgG. (FIG. 2) These results indicate that themembrane binding assay determines, at least in part, the binding ofradiolabeled secretase inhibitors to presenilin 1 fragments.

Subsequent experiments established that the lack of polypeptide B in theinitial immunoprecipitation experiments was due to aggregation uponboiling of the sample in reduced SDS sample buffer, indicating that allspecifically labeled polypeptides (A to D) can be specificallyimmunoprecipitated with antibodies to presenilin 1 under non-denaturingconditions.

FIG. 2 illustrates a fluorography of a 12% SDS-PAGE. The relativemobility of molecular weight standards (in kD) is indicated to the left.THP-1 membranes were incubated (30 minutes; room temperature) withExample 43T (30 nM) alone (panel A) or Example 43T (30 nM) in thepresence of Example 98 (panel B). The membranes were photolysed at 365nm for 30 minutes and the membranes harvested by centrifugation. Themembranes were extracted with 50 mM Tris, pH 7.5 containing 100 mM KCl,2 mM EDTA, 2% CHAPS in the presence of protease inhibitors for 1 hour at4° C. The membrane extracts were either directly fractionated bySDS-PAGE (lanes 1 and 6) or after immunoprecipitation with preimmune IgG(lanes 2 and 4) or antibodies to human presenilin 1 (lanes 3 and 5).Note the immunoprecipitation of specifically labeled bands ofapproximately 30, 20, and 10 kD after cross-linking in the absence ofExample 98, but not in the presence of Example 98. The higher molecularweight bands may represent the presenilin 1 holoprotein and/orpresenilin 1 aggregates formed in the presence of SDS.

Example 105

Purification of Cross-Linked Polypeptides by Affinity Chromatography

THP-1 membranes were prepared and cross-linked as in Example 104. Themembranes were extracted as in Example 104 at a protein concentration of10 mg membrane protein/1 ml extraction buffer. Normal mouse IgG (Sigma)or monoclonal antibody to the C-terminal loop of presenilin 1 wasimmobilized on agarose beads at 2 mg IgG per 1 ml of beads. The membraneextract was diluted one half with water and applied to a normal mouseIgG precolumn, followed by anti-presenilin 1 IgG. The column materialwas extensively washed with one half diluted extraction buffer, one halfdiluted extraction buffer containing 1M KCl, and eluted with 0.1 Mglycine, pH 2.5 in one half diluted extraction buffer. The resultingpolypeptides were analyzed by SDS-PAGE (12% acrylamide in the separatinggel), followed by fluorography (left top panel, FIG. 3), silver staining(right top panel, FIG. 3), immunoblotting using antibodies to theN-terminus of presenilin (left middle panel, FIG. 3) or the C-terminusof presenilin 1 (right middle panel, FIG. 3). In addition, the silverstain (right top panel, FIG. 3) was soaked in Amplify™ (Amersham),dried, and exposed the x-ray film. It is concluded that the specificallycross-linked bands A, B, and C can be enriched by presenilin 1 affinitychromatography. It should be noted that using an antibody to theN-terminus of presenilin 1, also band D could be enriched. PolypeptidesA and C are major silver stained protein bands containing thecross-linker of Example 11T and are immunoreactive with antibodies topresenilin 1. It should be noted that the extraction procedure used willnot dissociate the association of macromolecules in the presenilincomplex. Accordingly, one skilled in the art will understand that thistechnique can be employed to identify macromolecules associated with thebinding site that are involved in beta amyloid precursor processing.

FIG. 3 illustrates isolation of cross-linked polypeptides by presenilin1 affinity chromatography. THP-1 membranes were cross-linked as in FIG.2 and the resulting membrane extracts were applied to a normal mouse IgGSepharose, followed by an anti-presenilin 1 Sepharose. The startingmaterial (lanes 1), flow-through normal mouse IgG (lanes 2),flow-through presenilin 1 Sepharose (lanes 3), last wash prior toelution (lanes 4), and elution by lowering the pH (lanes 5) areindicated. The relative mobility of molecular weight markers isindicated. The left top panel shows a fluorography of a 12% SDS-PAGE.Note the enrichment of bands A to C on the presenilin 1 affinity column.The silver stain (top right panel) reveals that bands A and C areclearly enriched, distinguishable from contaminating proteins, andpresent. in purity sufficient for sequence analysis. The silver stainwas soaked in Amplify™ (Amersham), dried, and exposed to x-ray film(bottom left panel). It should be noted that major polypeptides in theelution fraction as revealed by silver staining perfectly align with theradioactivity as revealed by fluorography. The identify of band A aspresenilin 1 N-terminal fragments was revealed by immunoblotting usingN-terminal-specific antibodies (left middle panel), whereas band C wasidentified as presenilin C-terminal fragments (right middle panel).

It is understood by one skilled in the art that this or similarpurification schemes can be employed to isolate radiolabeled bindingpolypeptides in sufficient quantities to allow for N-terminal amino acidor mass spectoscropy analysis. Also, one skilled in the art understandsthat isolated radiolabeled polypeptides can be further fractionatedafter chemical or proteolytic digestion to isolate one or severalradiolabeled polypeptides in the sizes of approximately 2 to 100 aminoacids. Sequence analysis will reveal the location of the smallerpolypeptides in the protein sequence of the binding site molecules. Inaddition, this method can be used to define specifically cross-linkedamino acids in the binding site. This information can ultimately be usedin rational drug design for Alzheimer's disease. It should be noted thatboth N- and C-terminal presenilin 1 fragments are labeled by Example11T. This observation is consistent with the notion that the bindingsite is contained in proteolytic fragments of presenilin 1 generatedupon incorporation in the presenilin 1 complex.

Example 106

Evidence for the Involvement of Presenilin 2 in the Binding Site

THP-1 membranes were prepared and analyzed as in Example 104. Thepresenilin 1 antibodies were replaced with rabbit polyclonal antibodiesspecific for presenilin 2. The following modifications were included incomparison to Example 104: samples were pre-absorbed with normal rabbitIgG and protein A Sepharose was used instead of anti-mouse IgGSepharose. The resulting immunoprecipitates were analyzed by SDS-PAGE(12% acrylamide in the separating gel) followed by fluorography. (SeeFIG. 4) Bands A and B was specifically precipitated with an antibody topresenilin 2 (N-terminus), whereas antibodies to the C-terminus ofpresenilin 2 preferentially identified band B. These results indicatethat the membrane binding assay determines, at least in part, thebinding of radiolabeled secretase inhibitors to presenilin 2 fragments.One skilled in the art will realize that the cross-linking assay can beused to identify compounds with preferential affinity for eitherpresenilin 1 or 2. Membranes derived from organisms lacking eitherpresenilin 1 or 2, or both, might be used for the same purpose.

FIG. 4 illustrates a fluorography of a 12% SDS-PAGE. The relativemobility of molecular weight standards (in kD) is indicated to theright. THP-1 membranes were incubated (30 minutes; room temperature)with Example 11T, photolyzed at 365nm for 30 minutes, and the membranesharvested by centrifugation. The membranes were extracted with 50 mMTris, pH 7.5 containing 100 mM KCl, 2 mM EDTA, 2% CHAPS in the presenceof protease inhibitors for 1 hour at 4° C. The membrane extracts wereeither directly fractionated by SDS-PAGE (lane 1) or afterimmunoprecipitation with preimmune IgG (lane 4) or antibodies to humanpresenilin 2 (lane 2, PS-2 N-terminal specific antibody; lane 3, PS-2C-terminal specific antibody). Note the immunoprecipitation ofspecifically labeled bands A and B of approximately 30 and 25 kD.

It is understood by one skilled in the art that the assays disclosedherein, specifically the Radio Competition Binding Assay and thecross-linking Assay may be employed to differentiate between inhibitorsspecific for presenilin-1 and presenilin-2. For example, differentialcompetition for radioactivity incorporation in bands A to D wouldindicate presenilin-1 and/or presenilin-2 specific compounds. Moreover,binding to membranes derived from mammalian cells deficient in eitherPS-1 or PS-2 may be employed to identify PS-1 or PS-2 specificcompounds. For example these cells may be derived from organisms, forexample murine, which are gene targeted for PS-1 or PS-2. Examples ofcells include fibroblasts, neurons, and whole embryonic membranes.

It is understood that the isolation and sequence data for presenilin-1(PS-1) cloning has been published in Sherrington R et al., Nature, Vol375, pp754-760, 1995, herein incorporated by reference. It is alsounderstood that the isolation and sequence data for presenilin-2 (PS-2)cloning has been published in Rogaev E.I. et al., Nature, Vol 376,pp774-778, 1995, herein incorporated by reference.

In Vivo Diagnostic Imaging Utility

The radiolabeled compounds of the invention are useful asradiopharmaceuticals for imaging sites involved in beta-amyloidproduction, and thus may be used to diagnose present or potentialdisorders involving beta-amyloid production, including but not limitedto Alzheimer's disease. The patient may be any type of a mammal, but ispreferably a human. The radiolabeled compounds may be used alone, or maybe employed as a composition with a radiopharmaceutically acceptablecarrier, and/or in combination with other diagnostic or therapeuticagents. Suitable radiopharmaceuticals carriers and suitable amountsthereof are well known in the art, and can be found in, for example,Remington's Pharmaceutical Sciences, Gennaro, A. R., ed., MackPublishing Company, Easton, Pa. (1985), and The United StatesPharmacopia—The National Formulary, 22nd Revision, Mack PrintingCompany, Easton, Pa. (1990), standard reference texts in thepharmaceutical field. Other materials may be added, as convenient, tostabilize the composition, as those skilled in the art will recognize,including antioxidizing agents such as sodium bisulfite, sodium sulfite,ascorbic acid, gentisic acid or citric acid (or their salts) or sodiumethylenediamine tetraacetic acid (sodium EDTA), as is well known in theart. Such other materials, as well as suitable amounts thereof, are alsodescribed in Remington's Pharmaceutical Sciences and The United StatesPharmacopia—The National Formulary, cited above.

The present invention also includes radiopharmaceutical kits containingthe labeled compounds of the invention. Such kits may contain thelabeled compounds in sterile lyophilized form, and may include a sterilecontainer of a radiopharmaceutically acceptable reconstitution liquid.Suitable reconstitution liquids are disclosed in Remington'sPharmaceutical Sciences and The United States Pharmacopia—The NationalFormulary, cited above. Such kits may alternatively contain a sterilecontainer of a composition of the radiolabeled compounds of theinvention. Such kits may also include, if desired, other conventionalkit components, such as, for example, one or more carriers, one or moreadditional vials for mixing. Instructions, either as inserts or labels,indicating quantities of the labeled compounds of the invention andcarrier, guidelines for mixing these components, and protocols foradministration may also be included in the kit. Sterilization of thecontainers and any materials included in the kit and lyophilization(also referred to as freeze-drying) of the labeled compounds of theinvention may be carried out using conventional sterilization andlyophilization methodologies known to those skilled in the art.

To carry out the method of the invention, the radiolabeled compounds aregenerally administered intravenously, by bolus injection, although theymay be administered by any means that produces contact of the compoundswith sites of beta-amyloid production, particularly in sites in thebrain. Suitable amounts for administration will be readily ascertainableto those skilled in the art, once armed with the present disclosure. Thedosage administered will, of course, vary depending up such knownfactors as the particular compound administered, the age, health andweight or the nature and extent of any symptoms experienced by thepatient, the amount of radiolabeling, the particular radionuclide usedas the label, the rate of clearance of the radiolabeled compounds fromthe patient.

Acceptable ranges for administration of radiolabeled materials aretabulated, for example, in the Physicians Desk Reference (PDR) forNuclear Medicine, published by Medical Exonomics Company, a well-knownreference text. A discussion of some of the aforementionedconsiderations is provided in Eckelman et al., J. Nucl. Med., Vol. 209,pp. 350-357 (1979). By way of general guidance, a dosage range of theradiolabeled compounds of the invention may be between about 1 and about40 mCi.

Once the radiolabeled compounds of the invention are administered, thepresence of sites involved in beta-amyloid production may be visualizedusing standard imaging systems. Such imaging systems are well known inthe art, and are discussed, for example, in Macovski, A., MedicalImaging Systems, Information and Systems Science Series, Kailath, T.,ed., Prentice-Hall, Inc., Englewood Cliffs, N.J. (1983). Particularlypreferred is positron emission tomography (PET). Specifically, imagingis carried out by scanning the entire patient, or a particular region ofthe patient using the detection system, and detecting the radioisotopesignal. The detected signal is then converted into an image. Theresultant images should be read by an experienced observer, such as, forexample, a nuclear medicine physician. The foregoing process is referredto herein as “imaging” the patient. Generally, imaging is carried outabout 1 minute to about 48 hours following administration of theradiolabeled compound of the invention. The precise timing of theimaging will be dependant upon such factors as the half-life of theradioisotope employed, and the clearance rate of the compoundadministered, as will be readily apparent to those skilled in the art.Preferably, imaging is carried out between about 1 minute and about 4hours following administration.

The advantage of employing the radiolabeled compounds of the invention,which have the ability to localize specifically and with high affinityin sites involved in beta-amyloid production, to detect the presence ofsuch sites involved in beta-amyloid production and/or to diagnosedisorders in a patient involving beta-amyloid production, will bereadily apparent to those skilled in the art, once armed with thepresent disclosure.

Dosage and Formulation

The compounds determined from the present invention can be administeredorally using any pharmaceutically acceptable dosage form known in theart for such administration. The active ingredient can be supplied insolid dosage forms such as dry powders, granules, tablets or capsules,or in liquid dosage forms, such as syrups or aqueous suspensions. Theactive ingredient can be administered alone, but is generallyadministered with a pharmaceutical carrier. A valuable treatise withrespect to pharmaceutical dosage forms is Remington's PharmaceuticalSciences, Mack Publishing.

The compounds determined from the present invention can be administeredin such oral dosage forms as tablets, capsules (each of which includessustained release or timed release formulations), pills, powders,granules, elixirs, tinctures, suspensions, syrups, and emulsions.Likewise, they may also be administered in intravenous (bolus orinfusion), intraperitoneal, subcutaneous, or intramuscular form, allusing dosage forms well known to those of ordinary skill in thepharmaceutical arts. An effective but non-toxic amount of the compounddesired can be employed to prevent or treat neurological disordersrelated to β-amyloid production or accumulation, such as Alzheimer'sdisease and Down's Syndrome.

The compounds of this invention can be administered by any means thatproduces contact of the active agent with the agent's site of action inthe body of a host, such as a human or a mammal. They can beadministered by any conventional means available for use in conjunctionwith pharmaceuticals, either as individual therapeutic agents or in acombination of therapeutic agents. They can be administered alone, butgenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice.

The dosage regimen for the compounds determined from the presentinvention will, of course, vary depending upon known factors, such asthe pharmacodynamic characteristics of the particular agent and its modeand route of administration; the species, age, sex, health, medicalcondition, and weight of the recipient; the nature and extent of thesymptoms; the kind of concurrent treatment; the frequency of treatment;the route of administration, the renal and hepatic function of thepatient,and the effect desired. An ordinarily skilled physician orveterinarian can readily determine and prescribe the effective amount ofthe drug required to prevent, counter, or arrest the progress of thecondition.

Advantageously, compounds determined from the present invention may beadministered in a single daily dose, or the total daily dosage may beadministered in divided doses of two, three, or four times daily.

The compounds identified using the present invention can be administeredin intranasal form via topical use of suitable intranasal vehicles, orvia transdermal routes, using those forms of transdermal skin patcheswall known to those of ordinary skill in that art. To be administered inthe form of a transdermal delivery system, the dosage administrationwill, of course, be continuous rather than intermittant throughout thedosage regimen.

In the methods of the present invention, the compounds herein describedin detail can form the active ingredient, and are typically administeredin admixture with suitable pharmaceutical diluents, excipients, orcarriers (collectively referred to herein as carrier materials) suitablyselected with respect to the intended form of administration, that is,oral tablets, capsules, elixirs, syrups and the like, and consistentwith conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic, pharmaceutically acceptable, inert carrier such as lactose,starch, sucrose, glucose, methyl callulose, magnesium stearate,dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like;for oral administration in liquid form, the oral drug components can becombined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Moreover, whendesired or necessary, suitable binders, lubricants, disintegratingagents, and coloring agents can also be incorporated into the mixture.Suitable binders include starch, gelatin, natural sugars such as glucoseor β-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes, and the like. Lubricants used in thesedosage forms include sodium oleate, sodium stearate, magnesium stearate,sodium benzoate, sodium acetate, sodium chloride, and the like.Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthan gum, and the like.

The compounds determined from the present invention can also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamallar vesicles, and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine, or phosphatidylcholines.

Compounds of the present invention may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compoundsdetermined from the present invention may be coupled to a class ofbiodegradable polymers useful in achieving controlled release of a drug,for example, polylactic acid, polyglycolic acid, copolymers ofpolylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans,polycyanoacylates, and crosslinked or amphipathic block copolymers ofhydrogels.

Gelatin capsules may contain the active ingredient and powderedcarriers, such as lactose, starch, cellulose derivatives, magnesiumstearate, stearic acid, and the like. Similar diluents can be used tomake compressed tablets. Both tablets and capsules can be manufacturedas sustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance. In general, water, a suitableoil, saline, aqueous dextrose (glucose), and related sugar solutions andglycols such as propylene glycol or polyethylene glycols are suitablecarriers for parenteral solutions. Solutions for parenteraladministration preferably contain a water soluble salt of the activeingredient, suitable stabilizing agents, and if necessary, buffersubstances. Antioxidizing agents such as sodium bisulfite, sodiumsulfite, or ascorbic acid, either alone or combined, are suitablestabilizing agents. Also used are citric acid and its salts and sodiumEDTA. In addition, parenteral solutions can contain preservatives, suchas benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

1. A method of screening for inhibitors of beta-amyloid productioncomprising, 1) contacting a potential inhibitor of beta-amyloidproduction and a tagged inhibitor of beta-amyloid production with atleast one macromolecule involved in the processing of APP and theproduction of beta-amyloid peptide, said macromolecule containing abinding site specific for said tagged inhibitor of beta-amyloidproduction; 2) separating the tagged inhibitor of beta-amyloidproduction bound to said macromolecule from the tagged inhibitor ofbeta-amyloid production free from said macromolecule; and 3) determiningan inhibitory concentration of the potential inhibitor of beta-amyloidproduction from the concentration of tagged inhibitor of beta-amyloidproduction bound to said macromolecule.
 2. The method of claim 1 whereinthe tagged inhibitor of beta-amyloid production comprises a radiolabeledinhibitor of beta-amyloid production, a fluorescence labeled inhibitorof beta-amyloid production or a biotin labeled inhibitor of beta-amyloidproduction.
 3. The method of claim 1 wherein the tagged inhibitor ofbeta-amyloid production comprises a radiolabeled inhibitor ofbeta-amyloid production.
 4. The method of claim 1 wherein the taggedinhibitor of beta-amyloid production comprises a tritium or iodineradiolabeled inhibitor of beta-amyloid production.
 5. The method ofclaim 1 wherein the tagged inhibitor of beta-amyloid productioncomprises a tritium radiolabeled inhibitor of beta-amyloid production.6. The method of claim 1 wherein the tagged inhibitor of beta-amyloidproduction comprises a compound of the Formula (I):

wherein: at least one atom of the compound of the Formula (I) isradiolabeled; Q is NH₂; R³ is C₁-C₆ alkyl substituted with 0-1 R⁴; R⁴ isH, OH, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₁₀ carbocycle,C₆-C₁₀ aryl, or 5 to 10 membered heterocycle; R⁵ is H, OR¹⁴; C₁-C₆ alkylsubstituted with 0-3 R^(5b); C₁-C₆ alkoxy substituted with 0-3 R^(5b);C₂-C₆ alkenyl substituted with 0-3 R^(5b); C₂-C₆ alkynyl substitutedwith 0-3 R^(5b); C₃-C₁₀ carbocycle substituted with 0-3 R^(5c); C₆-C₁₀aryl substituted with 0-3 R^(5c); or 5 to 10 membered heterocyclesubstituted with 0-3 R^(5c); R^(5b), at each occurrence, isindependently selected from: H, C₁-C₆ alkyl, CF₃, OR¹⁴, Cl, F, Br, I,═O, CN, NO₂, NR¹⁵R¹⁶; C₃-C₁₀ carbocycle substituted with 0-3 R^(5c);C₆-C₁₀ aryl substituted with 0-3 R^(5c); or 5 to 10 membered heterocyclesubstituted with 0-3 R^(5c); R^(5c), at each occurrence, isindependently selected from H, OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br,I, CN, NO₂, NR¹⁵R¹⁶, or CF₃; R⁶ is H; C₁-C₆ alkyl substituted with 0-3R^(6a); C₃-C₁₀ carbocycle substituted with 0-3 R^(6b); or C₆-C₁₀ arylsubstituted with 0-3R^(6b); R^(6a), at each occurrence, is independentlyselected from H, C₁-C₆ alkyl, OR¹⁴, Cl, F, Br, I, ═O, CN, NO₂, NR¹⁵R¹⁶,phenyl or CF₃; R^(6b), at each occurrence, is independently selectedfrom H, OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,or CF₃; W is —(CR⁸R^(8a))_(p)—; p is 0 to 4; R⁸ and R^(8a), at eachoccurrence, are independently selected from H, C₁-C₄ alkyl, C₂-C₄alkenyl, C₂-C₄ alkynyl and C₃-C₈ cycloalkyl; X is a bond; C₆-C₁₀ arylsubstituted with 0-3 R^(Xb); C₃-C₁₀ carbocycle substituted with 0-3R^(Xb); or 5 to 10 membered heterocycle substituted with 0-3 R^(Xb);R^(Xb), at each occurrence, is independently selected from H, OH, C₁-C₆alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶, or CF₃; Y is a bondor —(CR⁹R^(9a))t-V—(CR⁹R⁹a)_(u)u; t is 0 to 3; u is 0 to 3; R⁹ andR^(9a), at each occurrence, are independently selected from H, C₁-C₆alkyl or C₃-C₈ cycloalkyl; V is a bond, —C(═O)—, —O—, —S—, —S(═O)—,—S(═O)₂—, —N(R¹⁹)—, —C(═O)NR^(19b)—, NR^(19b)C(═O)—, —NR^(19b)S(═O)₂—,—S(═O)₂NR^(19b)—, —NR^(19b)S(═O)—, —S(═O)NR^(19b)—, —C(═O)O—, or—OC(═O)—; Z is H; C₁-C₈ alkyl substituted with 0-2 R¹²; C₂-C₄ alkenylsubstituted with 0-2 R¹²; C₂-C₄ alkynyl substituted with 0-2 R¹²; C₆-C₁₀aryl substituted with 0-4 R^(12b); C₃-C₁₀ carbocycle substituted with0-4 R^(12b); or 5 to 10 membered heterocycle substituted with 0-3R^(12b); R¹² is C₆-C₁₀ aryl substituted with 0-4 R^(12b); C₃-C₁₀carbocycle substituted with 0-4 R^(12b); or 5 to 10 membered heterocyclesubstituted with 0-3 R^(12b); R^(12b), at each occurrence, isindependently selected from H, OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br,I, CN, NO₂, NR¹⁵R¹⁶, or CF₃; B is a 5 to 10 membered lactam, wherein thelactam is saturated, partially saturated or unsaturated; wherein eachadditional lactam carbon is substituted with 0-2 R¹¹; and, optionally,the lactam contains a heteroatom selected from —O—, —S—, —S(═O)—,—S(═O)₂—, —N═, and —N(R¹⁰)—; R¹⁰ is H, C(═O)R¹⁷, C(═O)OR¹⁷,C(═O)NR¹⁸R¹⁹, S(═O)₂NR¹⁸R¹⁹, S(═O)₂R¹⁷; C₁-C₆ alkyl optionallysubstituted with R^(10a); C₆-C₁₀ aryl substituted with 0-4 R^(10b);C₃-C₁₀ carbocycle substituted with 0-3 R^(10b); or 5 to 10 memberedheterocycle optionally substituted with 0-3 R^(10b); R^(10a), at eachoccurrence, is independently selected from H, C₁-C₆ alkyl, C₃-C₆cycloalkyl, OR¹⁴, Cl, F, Br, I, ═O, CN, NO₂, NR¹⁵R¹⁶, phenyl or CF₃;R^(10b), at each occurrence, is independently selected from H, OH, C₁-C₆alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶, or CF₃; R¹¹ isC₁-C₄ alkoxy, Cl, F, Br, I, ═O, CN, NO₂, NR¹⁸R¹⁹, C(═O)R¹⁷, C(═O)OR¹⁷,C(═O)NR¹⁸R¹⁹, S(═O)₂NR¹⁸R¹⁹, CF₃; C₁-C₆ alkyl optionally substitutedwith R^(11a); C₆-C₁₀ aryl substituted with 0-3 R^(11b); C₃-C¹⁰carbocycle substituted with 0-3 R^(11b); or 5 to 10 membered heterocyclesubstituted with 0-3 R^(11b); alternatively, two R¹¹ substituents on thesame carbon atoms may be combined to form a C₃-C₆ carbocycle;alternatively, two R¹¹ substituents on adjacent carbon atoms may becombined to form a C₃-C₆ carbocycle or a benzo fused radical, whereinsaid benzo fused radical is substituted with 0-3 R¹³; R^(11a), at eachoccurrence, is independently selected from H, C₁-C₆ alkyl, OR¹⁴, Cl, F,Br, I, ═O, CN, NO₂, NR¹⁵R^(l6), phenyl or CF₃; R^(11b), at eachoccurrence, is independently selected from H, OH, C₁-C₆ alkyl, C₁-C₄alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶, or CF₃; R¹³, at each occurrence,is independently selected from H, OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F,Br, I, CN, NO₂, NR¹⁵R¹⁶, or CF₃; R¹⁴ is H, phenyl, benzyl, C₁-C₆ alkyl,or C₂-C₆ alkoxyalkyl; R¹⁵, at each occurrence, is independently selectedfrom H, C₁-C₆ alkyl, benzyl, phenethyl, —C(═O)—(C₁-C₆ alkyl) and—S(═O)₂—(C₁-C₆ alkyl); R¹⁶, at each occurrence, is independentlyselected from H, OH, C₁-C₆ alkyl, benzyl, phenethyl, —C(═O)—(C₁-C₆alkyl) and —S(═O)₂—(C₁-C₆ alkyl); R¹⁷ is H, phenyl, benzyl, C₁-C₆ alkyl,or C₂-C₆ alkoxyalkyl; R¹⁸, at each occurrence, is independently selectedfrom H, C₁-C₆ alkyl, benzyl, phenethyl, —C(═O)—(C₁-C₆ alkyl) and—S(═O)₂—(C₁-C₆ alkyl); and R¹⁹, at each occurrence, is independentlyselected from H, OH, C₁-C₆ alkyl, phenyl, benzyl, phenethyl,—C(═O)—(C₁-C₆ alkyl) and —S(═O)₂—(C₁-C₆ alkyl); R^(19b) is H, C₁-C₆alkyl, C₃-C₈ cycloalkyl, phenyl, benzyl or phenethyl; and R²⁰ is H orC₁-C₆ alkyl.
 7. The method of claim 6 wherein R³ is C₃-C₆ alkyl.
 8. Themethod of claim 6 wherein R³ is C₃-C₆ alkyl substituted with about 1 toabout 4 ³H.
 9. The method of claim 6 wherein the tagged inhibitor ofbeta-amyloid production comprises a compound of the Formula (II):

wherein: at least one atom of the compound of the Formula (II) isradiolabeled.
 10. The method of claim 9 wherein R³ is C₃-C₆ alkylsubstituted with about 1 to about 4 ³H.
 11. The method of claim 1wherein the tagged inhibitor of beta-amyloid production comprises acompound of Formula:

wherein m is about
 2. 12. The method of claim 1 wherein the taggedinhibitor of beta-amyloid production comprises a compound of Formula(I-43T):

wherein m is about
 2. 13. The method of claim 1 wherein at least onemacromolecule involved in the processing of APP and the production ofbeta-amyloid peptide comprises presenilin 1 or a fragment ofpresenilin
 1. 14. The method of claim 1 wherein at least onemacromolecule involved in the processing of APP and/or the production ofbeta-amyloid peptide comprises comprises: (1) presenilin-1; (2)presenilin-2; (3) β secretase; (4) α secretase; (5) γ secretase; or (6)BACE/memapsin 2; or any fragment or derivative thereof.
 15. The methodof claim 1 wherein the inhibitory concentration is half maximalinhibitory concentration.
 16. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a therapeutically effectiveamount of an inhibitor of beta-amyloid production identified by thescreening assay of claim 1 or a pharmaceutically acceptable salt orprodrug form thereof.
 17. A method for treating degenerativeneurological disorders involving beta-amyloid production comprisingadministering to a host in need of such treatment a therapeuticallyeffective amount of an inhibitor of beta-amyloid production identifiedby the screening assay of claim 1 or a pharmaceutically acceptable saltor prodrug form thereof.
 18. A method for treating Alzheimer's diseasecomprising administering to a host in need of such treatment atherapeutically effective amount of an inhibitor of beta-amyloidproduction identified by the screening assay of claim 1 or apharmaceutically acceptable salt or prodrug form thereof.
 19. A methodof identifying a macromolecule involved in APP processing comprising 1)contacting a tagged inhibitor of beta-amyloid production with materialsuspected to contain a macromolecule involved in APP processing; 2)separating a complex comprising a tagged inhibitor of beta-amyloidproduction and a macromolecule involved in APP processing; and 3)identifying the complex.
 20. The method of claim 19 wherein the taggedinhibitor of beta-amyloid production comprises a radiolabeled inhibitorof beta-amyloid production, a fluorescence labeled inhibitor ofbeta-amyloid production, a biotin labeled inhibitor of beta-amyloidproduction, a photoaffinity labeled inhibitor of beta-amyloidproduction, or any combination of tags thereof in one inhibitor ofbeta-amyloid production.
 21. The method of claim 19 wherein the taggedinhibitor of beta-amyloid production comprises a radiolabeled inhibitorof beta-amyloid production.
 22. The method of claim 19 wherein thetagged inhibitor of beta-amyloid production comprises a tritium labeledinhibitor of beta-amyloid production.
 23. The method of claim 19 whereinthe tagged inhibitor of beta-amyloid production comprises a compound ofFormula (I):

wherein: at least one atom of the compound of the Formula (I) isradiolabeled; Q is NH₂; R³ is C₁-C₆ alkyl substituted with 0-1 R⁴; R⁴ isH, OH, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₁₀ carbocycle,C₆-C₁₀ aryl, or 5 to 10 membered heterocycle; R⁵ is H, OR¹⁴; C₁-C₆ alkylsubstituted with 0-3 R^(5b); C₁-C₆ alkoxy substituted with 0-3 R^(5b);C₂-C₆ alkenyl substituted with 0-3 R^(5b); C₂-C₆ alkynyl substitutedwith 0-3 R^(5b); C₃-C₁₀ carbocycle substituted with 0-3 R^(5c); C₆-C₁₀aryl substituted with 0-3 R^(5c); or 5 to 10 membered heterocyclesubstituted with 0-3 R^(5c); R^(5b), at each occurrence, isindependently selected from: H, C₁-C₆ alkyl, CF₃, OR¹⁴, Cl, F, Br, I,═O, CN, NO₂, NR¹⁵R¹⁶; C₃-C₁₀ carbocycle substituted with 0-3 R^(5c);C₆-C₁₀ aryl substituted with 0-3 R^(5c); or 5 to 10 membered heterocyclesubstituted with 0-3 R^(5c); R^(5c), at each occurrence, isindependently selected from H, OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br,I, CN, NO₂, NR¹⁵R¹⁶, or CF₃; R⁶ is H; C₁-C₆ alkyl substituted with 0-3R^(6a); C₃-C₁₀ carbocycle substituted with 0-3 R^(6b); or C₆-C₁₀ arylsubstituted with 0-3R^(6b); R^(6a), at each occurrence, is independentlyselected from H, C₁-C₆ alkyl, OR¹⁴, Cl, F, Br, I, ═O, CN, NO₂, NR¹⁵R¹⁶,phenyl or CF₃; R^(6b), at each occurrence, is independently selectedfrom H, OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶,or CF₃; W is —(CR⁸R^(8a))_(p)—; p is 0 to 4; R⁸ and R^(8a), at eachoccurrence, are independently selected from H, C₁-C₄ alkyl, C₂-C₄alkenyl, C₂-C₄ alkynyl and C₃-C₈ cycloalkyl; X is a bond; C₆-C₁₀ arylsubstituted with 0-3 R^(Xb); C₃-C₁₀ carbocycle substituted with 0-3R^(Xb); or 5 to 10 membered heterocycle substituted with 0-3 R^(Xb);R^(Xb), at each occurrence, is independently selected from H, OH, C₁-C₆alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶, or CF₃; Y is a bondor —(CR⁹R^(9a))t-V—(CR⁹R^(9a))_(u)—; t is 0 to 3; u is 0 to 3; R⁹ andR^(9a), at each occurrence, are independently selected from H, C₁-C₆alkyl or C₃-C₈ cycloalkyl; V is a bond, —C(═O)—, —O—, —S—, —S(═O)—,—S(═O)₂—, —N(R¹⁹)—, —C(═O)NR^(19b)—, —NR^(19b)C(═O)—, —NR^(19b)S(═O)₂—,—S(═O)₂NR^(19b)—, —NR^(19b)S(═O)—, —S(═O)NR^(19b)—, —C(═O)O—, or—OC(═O)—; Z is H; C₁-C₈ alkyl substituted with 0-2 R^(12b); C₂-C₄alkenyl substituted with 0-2 R^(12b); C₂-C₄ alkynyl substituted with 0-2R^(12b); C₆-C₁₀ aryl substituted with 0-4 R^(12b); C₃-C₁₀ carbocyclesubstituted with 0-4 R^(12b); or 5 to 10 membered heterocyclesubstituted with 0-3 R^(12b); R¹² is C₆-C₁₀ aryl substituted with 0-4R^(12b); C₃-C₁₀ carbocycle substituted with 0-4 R^(12b); or 5 to 10membered heterocycle substituted with 0-3 R^(12b); R^(12b), at eachoccurrence, is independently selected from H, OH, C₁-C₆ alkyl, C₁-C₄alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶, or CF₃; B is a 5 to 10 memberedlactam, wherein the lactam is saturated, partially saturated orunsaturated; wherein each additional lactam carbon is substituted with0-2 R¹¹; and, optionally, the lactam contains a heteroatom selected from—O—, —S—, —S(═O)—, —S(═O)₂—, —N═ and —N(R¹⁰)—; R¹⁰ is H, C(═O)R¹⁷,C(═O)OR¹⁷, C(═O)NR¹⁸R¹⁹, S(═O)₂NR¹⁸R¹⁹, S(═O)₂R¹⁷; C₁-C₆ alkyloptionally substituted with R^(10a); C₆-C₁₀ aryl substituted with 0-4R^(10b); C₃-C₁₀ carbocycle substituted with 0-3 R^(10b); or 5 to 10membered heterocycle optionally substituted with 0-3 R^(10b); R^(10a),at each occurrence, is independently selected from H, C₁-C₆ alkyl, C₃-C₆cycloalkyl, OR¹⁴, Cl, F, Br, I, ═O, CN, NO₂, NR¹⁵R¹⁶, phenyl or CF₃;R^(10b), at each occurrence, is independently selected from H, OH, C₁-C₆alkyl, C₁-C₄ alkoxy, Cl, F, Br, I, CN, NO₂, NR¹⁵R¹⁶, or CF₃; R¹¹ isC₁-C₄ alkoxy, Cl, F, Br, I, ═O, CN, NO₂, NR¹⁸R¹⁹, C(═O)R¹⁷, C(═O)OR¹⁷,C(═O)NR¹⁸R¹⁹, S(═O)₂NR¹⁸R¹⁹, CF₃; C₁-C₆ alkyl optionally substitutedwith R^(11a); C₆-C₁₀ aryl substituted with 0-3 R^(11b); C₃-C₁₀carbocycle substituted with 0-3 R^(11b); or 5 to 10 membered heterocyclesubstituted with 0-3 R^(11b); alternatively, two R¹¹ substituents on thesame carbon atoms may be combined to form a C₃-C₆ carbocycle;alternatively, two R¹¹ substituents on adjacent carbon atoms may becombined to form a C₃-C₆ carbocycle or a benzo fused radical, whereinsaid benzo fused radical is substituted with 0-3 R¹³; R^(11a), at eachoccurrence, is independently selected from H, C₁-C₆ alkyl, OR¹⁴, Cl, F,Br, I, ═O, CN, NO₂, NR¹⁵R¹⁶, phenyl or CF₃; R^(11b), at each occurrence,is independently selected from H, OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F,Br, I, CN, NO₂, NR¹⁵R¹⁶, or CF₃; R¹³, at each occurrence, isindependently selected from H, OH, C₁-C₆ alkyl, C₁-C₄ alkoxy, Cl, F, Br,I, CN, NO₂, NR¹⁵R¹⁶, or CF₃; R¹⁴ is H, phenyl, benzyl, C₁-C₆ alkyl, orC₂-C₆ alkoxyalkyl; R¹⁵, at each occurrence, is independently selectedfrom H, C₁-C₆ alkyl, benzyl, phenethyl, —C(═O)—(C₁-C₆ alkyl) and—S(═O)₂—(C₁-C₆ alkyl); R¹⁶, at each occurrence, is independentlyselected from H, OH, C₁-C₆ alkyl, benzyl, phenethyl, —C(═O)—(C₁-C₆alkyl) and —S(═O)₂—(C₁-C₆ alkyl); R¹⁷ is H, phenyl, benzyl, C₁-C₆ alkyl,or C₂-C₆ alkoxyalkyl; R¹⁸, at each occurrence, is independently selectedfrom H, C₁-C₆ alkyl, benzyl, phenethyl, —C(═O)—(C₁-C₆ alkyl) and—S(═O)₂—(C₁-C₆ alkyl); and R¹⁹, at each occurrence, is independentlyselected from H, OH, C₁-C₆ alkyl, phenyl, benzyl, phenethyl,—C(═O)—(C₁-C₆ alkyl) and —S(═O)₂—(C₁-C₆ alkyl); R^(19b) is H, C₁-C₆alkyl, C₃-C₈ cycloalkyl, phenyl, benzyl or phenethyl; and R²⁰ is H orC₁-C₆ alkyl.
 24. The method of claim 19 wherein the tagged inhibitor ofbeta-amyloid production comprises a compound of the Formula (I-7T):

wherein m is about
 2. 25. The method of claim 19 wherein the taggedinhibitor of beta-amyloid production comprises a compound of the Formula(I-43T), wherein m is about
 2. 26. The method of claim 20 wherein thetagged inhibitor of beta-amyloid production is radiolabeled andphotoaffinity labeled.
 27. The method of claim 20 wherein the taggedinhibitor of beta-amyloid production comprises a compound of the Formula(I-11T):

wherein m is about
 2. 28. A macromolecule involved in APP processingcomprising a macromolecule to which a tagged inhibitor of beta-amyloidproduction binds to specifically.
 29. A macromolecule of claim 28wherein the tagged inhibitor of beta-amyloid production comprises aradiolabeled inhibitor of beta-amyloid production, a fluorescencelabeled inhibitor of beta-amyloid production, a biotin labeled inhibitorof beta-amyloid production, a photoaffinity labeled inhibitor ofbeta-amyloid production, or any combination of tags thereof in oneinhibitor of beta-amyloid production.
 30. A macromolecule of claim 28wherein the tagged inhibitor of beta-amyloid production comprises aradiolabeled inhibitor of beta-amyloid production.
 31. A macromoleculeof claim 28 wherein the tagged inhibitor of beta-amyloid productioncomprises a compound of the Formula (I-7T):

wherein m is about
 2. 32. A macromolecule of claim 28 wherein the taggedinhibitor of beta-amyloid production comprises a compound of the Formula(I-11T):

wherein m is about
 2. 33. A macromolecule of claim 28 wherein the taggedinhibitor of beta-amyloid production comprises a compound of the Formula(I-43T):

wherein m is about
 2. 34. The macromolecule of claim 28 comprisingpresenilin 1 or a fragment of presenilin
 1. 35. The macromolecule ofclaim 28 comprising presenilin 2 or a fragment of presenilin
 2. 36. Aninhibitor of beta-amyloid production comprising a compound whichinteracts with a binding site on a macromolecule involved in theproduction of beta-amyloid peptide; wherein said binding site is aspecific binding site for a compound of Formula (I-7T) or (I-43T)wherein m is about
 2. 37. An inhibitor of beta-amyloid production ofclaim 36 wherein the macromolecule involved in the production ofbeta-amyloid peptide is presenilin 1 or a fragment of presenilin
 1. 38.An inhibitor of beta-amyloid production of claim 36 wherein themacromolecule involved in the production of beta-amyloid peptide ispresenilin 2 or a fragment of presenilin
 2. 39. An inhibitor ofbeta-amyloid production of claim 36 comprising a compound whichinteracts with a binding site on a macromolecule involved in theproduction of beta-amyloid peptide; wherein said binding site is aspecific binding site for a compound of Formula (I-7T) wherein m isabout 2; and the compound demonstrates a half maximal inhibitoryconcentration less than 10 micromolar for beta-amyloid production. 40.An inhibitor of beta-amyloid production of claim 36 comprising acompound which interacts with a binding site on presenilin 1 or afragment of presenilin 1; wherein said binding site is a specificbinding site for a compound of Formula (I-7T) wherein m is about 2; andthe compound demonstrates a half maximal inhibitory concentration lessthan 10 micromolar for beta-amyloid production.
 41. An inhibitor ofbeta-amyloid production of claim 36 comprising a compound whichinteracts with a binding site on a macromolecule involved in theproduction of beta-amyloid peptide; wherein said binding site is aspecific binding site for a compound of Formula (I-43T) wherein m isabout 2; and the compound demonstrates a half maximal inhibitoryconcentration less than 10 micromolar for beta-amyloid production. 42.An inhibitor of beta-amyloid production of claim 36 comprising acompound which interacts with a binding site on presenilin 1 or afragment of presenilin 1; wherein said binding site is a specificbinding site for a compound of Formula (I-43T) wherein m is about 2; andthe compound demonstrates a half maximal inhibitory concentration lessthan 10 micromolar for beta-amyloid production.
 43. A tagged inhibitorof beta-amyloid production comprising a tagged compound which interactswith a binding site on a macromolecule involved in the production ofbeta-amyloid peptide; wherein said binding site is a specific bindingsite for a compound of Formula (I-7T) or (I-43T) wherein m is about 2.44. A tagged inhibitor of beta-amyloid production of claim 43 whereinthe macromolecule involved in the production of beta-amyloid peptide ispresenilin 1 or a fragment of presenilin
 1. 45. A tagged inhibitor ofbeta-amyloid production of claim 43 wherein the macromolecule involvedin the production of beta-amyloid peptide is presenilin 2 or a fragmentof presenilin
 2. 46. A tagged inhibitor of beta-amyloid production ofclaim 43 comprising a tagged compound which interacts with a bindingsite on a macromolecule involved in the production of beta-amyloidpeptide; wherein said binding site is a specific binding site for acompound of Formula (I-7T) wherein m is about 2; and the tagged compounddemonstrates a half maximal inhibitory concentration less than 10micromolar for beta-amyloid production.
 47. A tagged inhibitor ofbeta-amyloid production of claim 43 comprising a tagged compound whichinteracts with a binding site on presenilin 1 or a fragment ofpresenilin 1; wherein said binding site is a specific binding site for acompound of Formula (I-7T) wherein m is about 2; and the tagged compounddemonstrates a half maximal inhibitory concentration less than 10micromolar for beta-amyloid production.
 48. A tagged inhibitor ofbeta-amyloid production of claim 43 comprising a tagged compound whichinteracts with a binding site on a macromolecule involved in theproduction of beta-amyloid peptide; wherein said binding site is aspecific binding site for a compound of Formula (I-43T) wherein m isabout 2; and the tagged compound demonstrates a half maximal inhibitoryconcentration less than 10 micromolar for beta-amyloid production.
 49. Atagged inhibitor of beta-amyloid production of claim 43 comprising atagged compound which interacts with a binding site on presenilin 1 or afragment of presenilin 1; wherein said binding site is a specificbinding site for a compound of Formula (I-43T) wherein m is about 2; andthe tagged compound demonstrates a half maximal inhibitory concentrationless than 10 micromolar for beta-amyloid production.
 50. A method ofidentifying inhibitors as therapeutics for neurological and otherdisorders involved in APP processing and beta-amyloid productioncomprising (1) contacting at least one macromolecule involved in APPprocessing and beta-amyloid production, which macromolecule a taggedinhibitor of beta-amyloid production binds to specifically, with apotential beta-amyloid inhibitor; and (2) determining the level ofinhibition of APP processing and beta-amyloid production.
 51. The methodof claim 50 wherein the macromolecule is a complex of macromolecules.52. The method of claim 50 wherein the macromolecule is presenilin 1 ora fragment of presenilin
 1. 53. The method of claim 50 wherein themacromolecule is presenilin 2 or a fragment of presenilin
 2. 54. Amethod of treating Alzheimer's disease comprising administering to ahost in need of such treatment a therapeutically effective amount of aninhibitor of beta-amyloid production, or a pharmaceutically acceptablesalt or prodrug form thereof, wherein said inhibitor of beta-amyloidproduction binds to a binding site on a macromolecule involved in theproduction of beta-amyloid peptide and effects a decrease in productionof beta-amyloid peptide; wherein said binding site is a specific bindingsite for a compound of Formula (I-7T) or (I-43T) wherein m is about 2.55. The method of claim 54 wherein the macromolecule comprisespresenilin-1, a fragment of presenilin-1, presenilin-2, or a fragment ofpresenilin-2.
 56. A method of claim 54 wherein the binding site is aspecific binding site for a compound of Formula (I-43T) wherein m isabout
 2. 57. The method of claim 56 wherein the macromolecule comprisespresenilin-1 or a fragment of presenilin-1.
 58. The method of claim 56wherein the macromolecule comprises presenilin-2 or a fragment ofpresenilin-2.
 59. A method of in vivo diagnostic imaging comprisingadministering to a subject a diagnostically effective amount of aradiolabeled inhibitor of beta-amyloid production.
 60. A method of claim59 wherein said method is used in the diagnosis of a neurologicaldisease which involves APP processing or elevated levels ofbeta-amyloid, or both.
 61. A method of claim 59 wherein said method isused in the diagnosis of Alzheimer's disease.
 62. A method of claim 59wherein the radiolabeled inhibitor is suitable for imaging of the brainof the subject.
 63. A method of claim 59 wherein the radiolabeledinhibitor is radiolabeled with one or more radioisotope selected from³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ¹²³I, ¹²⁵I, or ¹³¹I.
 64. A method of claim59 wherein the inhibitor of beta-amyloid production is a compoundselected from any compound disclosed in or within the scope of compoundsdisclosed in a reference selected from: (1) United States patent U.S.Pat. No. 5,703,129; (2) PCT application WO98/22441 (or its priority U.S.Ser. No. 08/755,444); (3) PCT application WO98/22433 (or its priorityU.S. Ser. No. 08/807,538); (4) PCT application WO98/22430 (or itspriority U.S. Ser. No. 08/754,895); (5) PCT application WO98/22493 (orits priority U.S. Ser. No. 08/755,334); (6) PCT application WO98/22494(or its priorities U.S. Ser. Nos. 08/808,528, 08/807,528 or 08/807,427);(7) PCT application WO98/28268 (or its priority U.S. Ser. No.08/780,025); (8) PCT application WO98/38177; (9) PCT applicationWO95/09838; (10) PCT application WO99/67221; (11) PCT applicationWO99/67220; (12) PCT application WO99/67219; (13) PCT applicationWO95/66934; (14) PCT application WO00/24392; or (15) Ghosh et al., JACS(2000) 122:3522-2523; or any compound which inhibits beta-amyloidproduction and binds competitively with any of the foregoing compoundsin any of the assays described in the Utility section hereof; all ofwhich foregoing references are hereby incorporated by reference in theirentirety.
 65. A method of claim 59 wherein the inhibitor of beta-amyloidproduction exhibits activity as an inhibitor in the method of any ofclaim
 1. 66. A method of claim 59 wherein the inhibitor of beta-amyloidproduction binds to a macromolecule which is capable of being identifiedby the method of any of claim
 19. 67. A method of claim 59 wherein theinhibitor of beta-amyloid production binds to a macromolecule of any ofclaim
 28. 68. A method of claim 59 wherein the inhibitor of beta-amyloidproduction is selected from an inhibitor of any of claim
 36. 69. Amethod of claim 59 wherein the radiolabeled inhibitor of beta-amyloidproduction is a radiolabeled tagged inhibitor of any of claims
 43. 70. Amethod of claim 59 wherein the inhibitor of beta-amyloid production isselected from: (1) an inhibitor of presenilin-1; (2) an inhibitor ofpresenilin-2; (3) an inhibitor of β secretase; (4) an inhibitor of αsecretase; (5) an inhibitor of γ secretase; or (6) an inhibitor ofBACE/memapsin
 2. 71. A pharmaceutical composition suitable for in vivodiagnostic imaging comprising a radiolabeled inhibitor of beta-amyloidproduction.
 72. A pharmaceutical composition of claim 71 wherein thecomposition is used in the diagnosis of a neurological disease whichinvolves APP processing or elevated levels of beta-amyloid, or both. 73.A pharmaceutical composition of claim 71 wherein the composition is usedin the diagnosis of Alzheimer's disease.
 74. A pharmaceuticalcomposition of claim 71 wherein the radiolabeled inhibitor is suitablefor imaging of the brain of the subject.
 75. A pharmaceuticalcomposition of claim 71 wherein the radiolabeled inhibitor isradiolabeled with one or more radioisotope selected from ³H, ¹¹C, ¹⁴C,¹⁸F, ³²P, ³⁵S, ¹²³I, ¹²⁵I, or ¹³¹I.
 76. A pharmaceutical composition ofclaim 71 wherein the inhibitor of beta-amyloid production is a compoundselected from any compound disclosed in or within the scope of compoundsdisclosed in a reference selected from: (1) United States patent U.S.Pat. No. 5,703,129; (2) PCT application WO98/22441 (or its priority U.S.Ser. No. 08/755,444); (3) PCT application WO98/22433 (or its priorityU.S. Ser. No. 08/807,538); (4) PCT application WO98/22430 (or itspriority U.S. Ser. No. 08/754,895); (5) PCT application WO98/22493 (orits priority U.S. Ser. No. 08/755,334); (6) PCT application WO98/22494(or its priorities U.S. Ser. Nos. 08/808,528, 08/807,528 or 08/807,427);(7) PCT application WO98/28268 (or its priority U.S. Ser. No.08/780,025); (8) PCT application WO98/38177; (9) PCT applicationWO95/09838; (10) PCT application WO99/67221; (11) PCT applicationWO99/67220; (12) PCT application WO99/67219; (13) PCT applicationWO95/66934; (14) PCT application WO00/24392; or (15) Ghosh et al., JACS(2000) 122:3522-2523; or any compound which inhibits beta-amyloidproduction and binds competitively with any of the foregoing compoundsin any of the assays described in the Utility section hereof; all ofwhich foregoing references are hereby incorporated by reference in theirentirety.
 75. A pharmaceutical composition of claim 71 wherein theinhibitor of beta-amyloid production is selected from: (1) an inhibitorof presenilin-1; (2) an inhibitor of presenilin-2; (3) an inhibitor of βsecretase; (4) an inhibitor of α secretase; (5) an inhibitor of γsecretase; or (6) an inhibitor of BACE/memapsin 2.